WO2015136255A1 - Combined inhibition of caspase-2 and caspase-6 in neuroprotection and axon regeneration - Google Patents

Abstract

The invention relates to the use of agents for the inhibition of caspase-2 and caspase-6 in the protection of central and peripheral neurons. In particular, the invention comprises an inhibitor of caspase-2 for use in the treatment of a disease, a disorder or an injury of the nervous system by combined, sequential or separate administration with an inhibitor of caspase-6. Further provided is a composition comprising an inhibitor of caspase-2 and an inhibitor of caspase-6.

Description

COMBINED INHIBITION OF CASPASE-2 AND CASPASE-6 IN NEUROPROTECTION AND AXON REGENERATION

FIELD AND BACKGROUND OF THE INVENTION

The invention, in some embodiments, relates to the use of agents for inhibition of caspase-2 and caspase-6 in protection of central and peripheral neurons, such as retinal ganglion cells (RGC) and dorsal root ganglion neurons (DRGN), from apoptosis and promotion of axon regeneration.

Unlike peripheral nervous system axons, central nervous system (CNS) axons fail to regenerate after injury. Many growth-limiting factors have been implicated, including apoptosis of axotomised neurons, intrinsic blocks to growth, limiting supplies of neurotrophic factors and the presence of an axon growth inhibitory environment in the CNS neuropil.

Optic nerve (ON) injury induces progressive RGC death (Villegas-Perez et al, 1993, Berkelaar et al, 1994, Kermer et al, 1998), so that 70-75% of RGC are lost within 7 days (Berry et al, 1996, Berry et al, 1999, Agudo et al, 2008, Ahmed et al, 2011, Vigneswara et al, 2012) and 80-90% by 28 days, primarily as a result of apoptosis (Garcia-Valenzuela et al, 1994, Rabacchi et al, 1994, Isenmann et al, 1997).

Caspases are a family of cysteine-dependent proteases well known for their orchestration of apoptosis, although they also have non-apoptotic roles. They are expressed as pro-caspases and activated by either proximity induced dimerization (initiator caspases) or proteolytic cleavage (effector caspases) (Pop and Salvesen, 2009). This enables active caspase enzymes to cleave regulatory proteins that play roles in DNA replication (Casciola-Rosen et al, 1994, Song et al, 1996), DNA repair (Lazebnik et al, 1994), cell survival signalling (Leist et al,

1997) , cytoskeletal reorganisation and cellular disassembly (Porter et al, 1997, Bokoch,

1998) .

Injured RGC exclusively express and cleave caspase-2 (Ahmed et al, 2011, Vigneswara et al, 2012) and suppression of caspase-2, by either a chemically stabilised caspase-2 siRNA (siCASP2) or a pharmacological inhibitor, significantly protects RGC from death for at least 14 days after injury (Ahmed et al, 201 1, Vigneswara et al, 2012). Despite significant RGC neuroprotection by both of these pharmacological strategies, RGC axon regeneration is unaffected, suggesting that different signalling pathways regulate neuron survival and axon regeneration. The present inventors have shown previously that the cues for post-injury RGC survival and axon regeneration, which can be mediated by inflammation, are different, since both retinal and ON inflammation promotes RGC survival but only retinal inflammation is RGC axogenic (Ahmed et al, 2010). RGC survival and axon regeneration require the activation of both survival and growth signalling pathways.

SUMMARY OF THE INVENTION

Some embodiments of the invention relate to use of agents for combined inhibition of caspase-2 and caspase-6 for use in the treatment of a disease, a disorder or an injury of the nervous system by combined, sequential or separate administration with an inhibitor of caspase-6.

Aspects and embodiments of the invention are described in the specification herein below and in the appended claims.

Some embodiments of the invention relate to the use of an inhibitor of caspase-2 and an inhibitor of caspase-6 in therapy.

According to an aspect of some embodiments of the invention, there is provided an inhibitor of caspase-2 for use in the treatment of a disease, a disorder or an injury of the nervous system by combined, sequential or separate administration with an inhibitor of caspase-6.

According to an aspect of some embodiments of the invention, there is provided a method of treating a disease, a disorder or an injury of the nervous system by combined, sequential or separate administration of an inhibitor of caspase-2 and an inhibitor of caspase-6.

In some embodiments, the nervous system is selected from the group consisting of the central nervous system (CNS) and the peripheral nervous system (PNS).

In some embodiments, the central nervous system (CNS) comprises the visual system and the audio-vestibular system.

In some embodiments, the disease, disorder or injury of the CNS is selected from the group consisting of an ocular disease; an ocular disorder or an ocular injury; a spinal cord disease, a spinal cord disorder or a spinal cord injury; a brain disease, a brain disorder or a brain injury; and a disease, disorder or injury of the audio-vestibular system, or a combination thereof. In some embodiments, the disease, disorder or injury of the CNS is selected from the group consisting of a neurodegenerative disease (such as an acute neurodegenerative disease or a chronic neurodegenerative disease) and a neurological disorder, or a combination thereof.

In some embodiments, the disease, disorder or injury of the CNS is selected from the group consisting of inflammation, neurotoxicity, oxidative stress, and traumatic damage due to a tumor growth, or a combination thereof.

In some embodiments, the disease, disorder or injury of the CNS is an ocular disease, an ocular disorder or an ocular injury.

In some embodiments, the ocular disease, ocular disorder or ocular injury is selected from the group consisting of neurodegeneration, inflammation, and oxidative stress, or a combination thereof.

In some embodiments, the ocular disease, ocular disorder or ocular injury is selected from the group consisting of glaucoma (including open angle glaucoma and angle closure glaucoma), and diabetic retinopathy (DR) or a combination thereof.

In some embodiments, the ocular disease, ocular disorder or ocular injury comprises an optic neuropathy selected from the group consisting of ischemic optic neuropathy (ION) (such as non-arteritic ischemic optic neuropathy (NAION), hereditary optic neuropathy (such asLeber's hereditary optic neuropathy (LHON)), metabolic optic neuropathy, neuropathy due to a toxic agent, neuropathy caused by adverse drug reactions and neuropathy caused by vitamin deficiency, or a combination thereof.

In some embodiments, the ocular disease, ocular disorder or ocular injury is selected from the group consisting of optic neuritis, retinal artery occlusion, central retinal vein occlusion, brunch retinal vein occlusion and optic nerve injury, or a combination thereof.

In some embodiments, the ocular disease, ocular disorder or ocular injury is selected from the group consisting of ischemic injury, ischemia-reperfusion injury, mechanical injury, injury or interruption of nerve fibers, physically damaged nerve, neurite damage, and a condition associated with lack of retrograde supply of neurotrophic factor, or a combination thereof.

In some embodiments, the treatment provides neuroprotection.

In some embodiments, the treatment provides axon regeneration of neuronal cells, such as axon regeneration of retinal neuronal cells or dorsal root ganglion neurons. In some embodiments, the treatment provides both neuroprotection and axon regeneration of neuronal cells.

In some embodiments, the neuronal cells are cells of the peripheral nervous system (PNS).

In some embodiments, the neuronal cells are cells of the central nervous system (CNS).

In some embodiments, the neuroprotection comprises ocular neuroprotection, such as audio- vestibular neuroprotection.

In some embodiments, the neuroprotection is selected from the group consisting of protection of neural cells from apoptosis, promoting survival of neural cells, increasing the number of neural cell neuritis, increasing neurite cell outgrowth, promoting retinal gliosis, promoting regeneration of neural cells and increasing or stimulation of neurotrophic factors in the nervous system, or a combination thereof.

In some embodiments, the neural cell comprises a ganglion cell, such as a ganglion cell selected from the group consisting of a retinal ganglion cell, a spiral ganglion cell, a vestibular ganglion cell, a dorsal root ganglion cell and a peripheral ganglion cell, or a combination thereof.

In some embodiments, the neural cell comprises a retinal neural cell, such as a photoreceptor cell, a bipolar cell, a ganglion cell, a horizontal cell and an amacrine cell, or a combination thereof.

In some embodiments, the neural cell comprises a dorsal root ganglion cell.

In some embodiments, retinal neural cell comprises a retinal ganglion cell (RGC).

In some embodiments, neural cell comprises a dorsal root ganglion cell (DRGC).

In some embodiments, the neuroprotection comprises protecting neurons from death. In some such embodiments, death of the neuron is associated with one or more of a disease or disorder, a surgery, ischemia, ischemia/reperfusion, physical/mechanical trauma, a chemical agent, an infectious agent, an immunologic reaction and a nutritional imbalance.

In some embodiments, the inhibitor of caspase-2 and the inhibitor of caspase-6 are independently configured for contacting the neuron.

According to an aspect of some embodiments disclosed herein, there is provided an inhibitor of caspase-2 for use in providing neuroprotection and axon regeneration of neuronal cells by combined, sequential or separate administration with an inhibitor of caspase-6. According to an aspect of some embodiments of the invention, there is provided a composition comprising an inhibitor of caspase-2 and an inhibitor of caspase-6.

In some embodiments, the inhibitor of caspase-2 and the inhibitor of caspase-6 are independently selected from the group consisting of a small organic molecule, a protein, an antibody or fragment thereof, a peptide, a polypeptide, a peptidomimetic and a nucleic acid molecule; or a salt or prodrug thereof, or a combination thereof.

In some embodiments, at least one of the inhibitor of caspase-2 and the inhibitor of caspase-6 is a nucleic acid molecule independently selected from the group consisting of a single stranded antisense nucleic acid (ssNA), a double-stranded NA (dsNA), a small interfering NA (siNA), a short hairpin NA (shNA), a micro RNA (miRNA), an aptamer, and a ribozyme, or a salt or prodrug thereof, or a combination thereof.

In some embodiments, each of the ssNA or the dsNA independently comprises one or more of a modified nucleotide, an unmodified nucleotide, a nucleotide analogue and an unconventional moiety, such as an RNA, a DNA, a TNA or an ANA. In some embodiments, the inhibitor of caspase-2 is a dsRNA.

In some embodiments, at least one of the inhibitor of caspase-2 and the inhibitor of caspase-6 is a double-stranded NA (dsNA) independently selected from an unmodified double-stranded NA (dsNA) or a chemically modified double-stranded NA (dsNA); or a salt or prodrug thereof.

The nucleotides can be selected from naturally occurring or synthetic modified bases. Naturally occurring bases include adenine, guanine, cytosine, thymine and uracil. Modified bases of nucleotides include inosine, xanthine, hypoxanthine, 2- aminoadenine, 6-methyl, 2- propyl and other alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4- thiouracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine, 8- thiolalkyl adenines, 8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanines, 8- amino guanine, 8-thiol guanine, 8-thioalkyl guanines, 8- hydroxyl guanine and other substituted guanines, other aza and deaza adenines, other aza and deaza guanines, 5- trifluoromethyl uracil and 5- trifluoro cytosine. Molecules comprising one or more abasic moiety (unconventional or pseudonucleotide) are encompassed by the present invention, as well as molecules comprising alternating RNA and DNA nucleotides.

In addition, analogues of polynucleotides can be prepared wherein the structure of one or more nucleotide is fundamentally altered and better suited as therapeutic or experimental reagents. An example of a nucleotide analog is a peptide nucleic acid (PNA) wherein the deoxyribose (or ribose) phosphate backbone in DNA (or RNA) is replaced with a polyamide backbone which is similar to that found in peptides. PNA analogs have been shown to be resistant to enzymatic degradation and to have extended lives in vivo and in vitro.

Possible modifications to the sugar residue are manifold and include 2'-0 alkyl, locked nucleic acid (LNA), glycol nucleic acid (GNA), threose nucleic acid (TNA), arabinoside, altritol (ANA) and other, 6-membered sugars including morpholinos, and cyclohexinyls. Further, said molecules may additionally contain modifications on the sugar, such as 2' alkyl, 2' fluoro, 2Ό allyl, 2'amine and 2'alkoxy. Additional sugar modifications are discussed herein.

In some embodiments, the inhibitor of caspase-2 comprises an antisense strand sequence 5' AGGAGUUCCACAUUCUGGC 3' (SEQ ID No. 1) and a sense strand sequence 5' GCCAGAAUGUGGAACUCCU 3' (SEQ ID No. 2).

In some embodiments, the antisense strand of the inhibitor of caspase-2 comprises 2'-0- methyl sugar modified ribonucleotides in positions (5'>3') 2, 4, 6, 8, 11, 13, 15, 17 and 19; and wherein the sense strand of the inhibitor of caspase-2 comprises an L-DNA (L- deoxycytidine) nucleotide in position 18 and an inverted deoxyabasic moiety covalently attached at the 5' terminal of the strand.

In some embodiments, the inhibitor of caspase-6 comprises caspase-6 dominant negative (C6DN). In some embodiments, C6DN is coupled to a peptide, for example a cell penetrating peptide such as Penetratin-1 (Penl).

The inhibitor of caspase-2 and the inhibitor of caspase-6 may be administered by any of the conventional routes of administration. The chemically modified dsRNA compounds are administered orally, subcutaneously or parenterally including intravenous, intraarterial, intramuscular, intraperitoneally, intraocular, intracoronary, transtympanic, transcorneal, transepithelial, transmembrane and intranasal administration as well as intrathecal and infusion techniques. Implants of the compounds are also useful.

In some embodiments, the inhibitor of caspase-2 is for use by concurrent administration with the inhibitor of caspase-6.

In some embodiments, the inhibitor of caspase-2 and the inhibitor of caspase-6 are administered in a single dosage form. In some embodiments, the inhibitor of caspase-2 and the inhibitor of caspase-6 are administered in separate dosage forms.

In some embodiments, the inhibitor of caspase-2 is for sequential administration with the inhibitor of caspase-6. In some such embodiments, the inhibitor of caspase-2 is administered prior to administration of the inhibitor of caspase-6. In other such embodiments, the the inhibitor of caspase-2 is administered subsequent to administration of the inhibitor of caspase-6.

In some embodiments, the inhibitor of caspase-2 and the inhibitor of caspase-6 comprise different portions of a single molecule.

In some embodiments, there is provided the use of the inhibitor of caspase-2 and the inhibitor of caspase-6 as described herein, for combined, sequential or separate administration with a neurotrophic factor(s), for example but not exclusively, a neurotrophic factor selected from the group consisting of ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3) and fibroblast growth factor-2 (FGF-2), or any combination thereof.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. In case of conflict, the specification, including definitions, takes precedence.

As used herein, the terms "comprising", "including", "having" and grammatical variants thereof are to be taken as specifying the stated features, integers, steps or components but do not preclude the addition of one or more additional features, integers, steps, components or groups thereof. These terms encompass the terms "consisting of and "consisting essentially of .

As used herein, the indefinite articles "a" and "an" mean "at least one" or "one or more" unless the context clearly dictates otherwise.

As used herein the term "inhibitor" refers to a compound, which is capable of reducing (partially or fully) the expression of a gene or the activity of the product of such gene to an extent sufficient to achieve a desired biological or physiological effect.

As used herein a "therapeutic composition" refers to a preparation of one or more of the active ingredients with other components such as pharmaceutically-acceptable carriers and excipients. The purpose of a therapeutic composition is to facilitate administration of an active ingredient to a subject.

The term "pharmaceutically acceptable carrier" refers to a carrier or a diluent that does not cause significant irritation to a subject and does not substantially abrogate the activity and properties of the administered active ingredients. An adjuvant is included under these phrases. The term "excipient" refers to an inert substance added to a therapeutic composition to further facilitate administration of an active ingredient.

The term "unconventional moiety" as used herein refers to abasic ribose moiety, an abasic deoxyribose moiety, a deoxyribonucleotide, a modified deoxyribonucleotide, a mirror nucleotide, a non-base pairing nucleotide analog and a nucleotide joined to an adjacent nucleotide by a 2'-5' internucleotide phosphate bond; bridged nucleic acids including LNA and ethylene bridged nucleic acids.

In the context of the present invention, a "mirror" nucleotide (also referred to as a spieglemer), is a nucleotide analog with reverse chirality to the naturally occurring or commonly employed nucleotide, i.e., a mirror image of the naturally occurring or commonly employed nucleotide. The mirror nucleotide is a ribonucleotide (L-RNA) or a deoxyribonucleotide (L-DNA) and may further comprise at least one sugar or base modification and/or a backbone modification, such as a phosphorothioate or phosphonate moiety. US Patent No. 6,602,858 discloses nucleic acid catalysts comprising at least one L- nucleotide substitution. Mirror nucleotide includes for example L-DNA (L- deoxyriboadenosine-3 '-phosphate (mirror dA); L-deoxyribocytidine-3 '-phosphate (mirror dC); L-deoxyriboguanosine-3 '-phosphate (mirror dG); L-deoxyribothymidine-3 '-phosphate (mirror dT) and L-RNA (L-riboadenosine-3 '-phosphate (mirror rA); L-ribocytidine-3'- phosphate (mirror rC); L-riboguanosine-3 '-phosphate (mirror rG); L-ribouracil-3 '-phosphate (mirror dU).

Therapeutic compositions used in implementing the teachings herein may be formulated using techniques with which one of average skill in the art is familiar in a conventional manner using one or more pharmaceutically-acceptable carriers comprising excipients and adjuvants, which facilitate processing of the active ingredients into a therapeutic composition and generally includes mixing an amount of the active ingredients with the other components. Suitable techniques are described in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference. For example, therapeutic compositions useful in implementing the teachings herein may be manufactured by one or more processes that are well known in the art, e.g., mixing, blending, homogenizing, dissolving, granulating, emulsifying, encapsulating, entrapping and lyophilizing processes.

Therapeutic compositions suitable for implementing the teachings herein include compositions comprising active ingredients in an amount effective to achieve the intended purpose (a therapeutically effective amount). Determination of a therapeutically effective amount is well within the capability of those skilled in the art, for example, is initially estimated from animal models.

BRIEF DESCRIPTION OF THE FIGURES

Some embodiments of the invention are described herein with reference to the accompanying figures. The description, together with the figures, makes apparent to a person having ordinary skill in the art how some embodiments of the invention may be practiced.

In the Figures:

FIGs. 1A-1F show dose-response relationship for Penl-C6DN. Optic nerves were crushed and the retinae treated with a range of doses of Penl-C6DN from 0-7μΜ C6DN at day 0, 7 and 14 after ONC. At 19d after ONC (FluoroGold) FG was injected into the proximal ON stump and allowed to retrogradely fill surviving RGC. 2 days later, animals were killed, retinae were harvested, wholemounts were made and the number of FG labelled RGC was quantified by image analysis. FIGs. 1A-1E are representative photomicrographs of FluoroGold (FG) labeled RGC. FIG. IF is a bar graph showing the dose-response relationship for Pen-C6DN concentration and RGC survival, showing increased RGC survival with increasing concentrations of Penl-C6DN up to 5μΜ. Scale bar = 50μιη;*Ρ<0.05, ***P<0.0001;

FIGs. 2A-2D show the effects of 5μΜ Penl-C6DN alone or in combination with siCASP2 on RGC survival. Optimal dose of Penl-C6DN was intravitreally delivered either alone or in combination with siCASP2 immediately after ONC and at 7 and 14 days after ONC. At 21 days, animals were killed and retinae were harvested either for western blot analysis or to count the number of FG-labelled RGC in retinal wholemounts. Western blot analysis (FIG. 2 A) and subsequent densitometry (FIG. 2B) show that suppression of caspase-6 using 5μΜ Penl-C6DN prevents cleavage of Lamin A/C while siCASP2 suppresses the levels of caspase-2 in treated eyes. FluoroGold labelling of RGC (FIGs. 2C and 2D0 demonstrate that Penl-C6DN protects nearly 50% of RGC from apoptosis while intravitreal delivery of either Penl+siCASP2 or Penl-C6DN+siCASP2 promoted >95% of RGC survival. GAPDH was used as a protein loading control. ***P<0.0001. Scale bar in C = 50 μιη.

FIGs. 3A-3G show the effects of intravitreal delivery of optimized Penl-C6DN alone or in combination on promotion of RGC axon regeneration. After ONC, optimised Penl-C6DN either alone or in combination with siCASP2 was intravitreally injected at 0, 7 and 14 days. Animals were killed at day 21, optic nerves dissected out and processed for immunohistochemistry. FIGs. 3A-3D show GAP-43 stained images of regenerating RGC axons after treatment with Penl alone (3 A), Penl-C6DN (3B), Penl+siCASP (3C) and Penl- C6DN+siCASP2 (3D). Inset shows high power magnification of boxed region in 3D. FIG. 3E is a bar graph showing quantification of RGC axons at different distances beyond the lesion epicentre of the ON. *P<0.0001. Scale bars = ΙΟΟμιη. Anterograde FIGs. 3F-3H show Rhodamine B labelling after treatment with Penl-C6DN+siCASP to confirm regenerating RGC axons in the distal ON stump (examples shown by arrowheads) immediately past the lesion site (3F), at ΙΟΟΟμπι (FG) and at 2000μπι (3H) from the lesion site.

FIGs. 4A-4P show the effects of intravitreal delivery of optimised Penl-C6DN alone or in combination with siCASP2 on activation of retinal astrocytes and Miiller cells and increase of CNTF in treated eyes. At 21 days after ONC and treatment with C6DN alone or in combination with siCASP2, eyes were processed for immunohistochemistry. FIGs. 4A-4L show representative images of the retinae of Penl, Penl-C6DN and Penl+siCASP2 treated eyes, demonstrating GFAP⁺ activated astrocytes and Miiller cells with associated CNTF. Scale bar = ΙΟΟμπι. FIG. 4M is a bar graph showing the effect of Penl-C6DN alone or in combination with siCASP2 on number of activated Miiller cells in the retina. Combined Penl-C6DN+siCASP2 treated eyes showed the highest numbers of activated astrocytes/Miiller cells and CNTF localisation in the retina. FIGs. 4N and 40 show immunolocalisation of CNTF in pIII-tubulin⁺ RGC in Penl (4N) and Penl-C6DN+siCASP2- treated retinae (40). Scale bar = 50μπι. FIG. 4P is a bar graph showing levels of CNTF in eyes treated with combined Penl-C6DN+siCASP2. ELISA detected increased levels of CNTF in eyes treated with combined Penl-C6DN+siCASP2. ***P<0.0001.

FIGs. 5A-5J show the effects of intravitreal delivery of optimised Penl-C6DN alone or in combination with siCASP2 on adult retinal cultures prepared 5 days after ONC. Intact retinal cultures did not contain GFAP⁺ glia (FIGs. 5 A, 51), while increasing numbers of GFAP⁺ glia were observed in retina dissociated after 5 days and untreated (FIGs. 5B, 51) or with intravitreal treatment of Pen 1 (FIGs. 5C, 51), Penl-C6DN (FIGs. 5D, 51), Penl-siCASP2 (FIGs. 5E, 51) and Penl-C6DN+siCASP2 (FIGs. 5F, 51). Retinal cell cultures prepared from Penl-C6DN+siCASP2-treated eyes that have also had suppression of CNTF signalling with either the MAB228 ^g/eye) (FIGs. 5G, 51) or the JAK/STAT pathway inhibitor AG490 (17mM/eye) (FIGs. 5H, 51), contain significantly reduced numbers of GFAP⁺ glia to the baseline levels observed after ONC or ONC+Penl treatment. The numbers of GFAP⁺ glia positively correlated with the levels of CNTF production in culture, while treatment with MAB228 or AG490 suppressed CNTF production to baseline levels. ***P<0.0001, Scale bar = 50μπι.

FIGs. 6A-6L show the effects of intravitreal delivery of optimised Penl-C6DN alone or in combination with siCASP2 on adult retinal cultures prepared 5 days after ONC on neurite outgrowth. FIGs. 6A-6H show neurite growth in culture. In untreated cultures (FIG. 6A) and cultures prepared 5 days after Penl vehicle treatment (FIG. 6B), few if any RGC grew neurites, while retinal cultures prepared from animals treated with Penl -CNTF (l ^g/eye) (FIG. 6C) increased the mean neurite length (FIG. 61), mean number of RGC with neurites (FIG. 6 J) and RGC survival (FIG. 6K). In retinal cultures prepared from eyes treated with Penl-C6DN and Penl-siCASP2 (FIGs. 6E, 6I-K), similarly increased mean neurite length and number of RGC with neurites were observed, while RGC survival was 55% and 98%, respectively. In retinal cultures prepared from animals treated with Penl-C6DN+siCASP2 (FIGs. 6F, 6I-K), significantly more neurite outgrowth was observed in terms of RGC neurite length and the number of RGC with neurites, while RGC neuroprotection was also 98%. However, simultaneous treatment with either MAB228 (FIGS. 6G, 6I-K) or AG490 (FIGs. 6H, 6I-K) in Penl-C6DN+siCASP2-treated cultures abrogated RGC neurite outgrowth without affecting RGC viability. ELISA from cells treatment with Penl-C6DN+siCASP2 (FIG. 6L) showed significantly high levels of CNTF compared to those treated with CNTF or other monotherapies. **P<0.001, ***P<0.0001, Scale bar = 40μιη.

FIGs. 7A-7H show in vivo that Penl-C6DN+siCASP2-stimulated RGC axon regeneration is abrogated by MAB228 and AG490, without affecting RGC survival. After ONC, animals were intravitreally injected with optimised Penl-C6DN+siCASP2 and rat IgG (5ug/eye), MAB228 (5ug/eye) and AG490 (17mM/eye). RGC survival was quantified by FG counting in retinal wholemounts and showed that blocking Penl-C6DN+siCASP2-mediated RGC axon growth by MAB228 or AG490 did not impact on RGC survival (FIGs. 7A-7D) but did block RGC axon regeneration (FIGs. 7E-7H). *P<0.0001 compared to Penl- C6DN+siCASP2+MAB228 or Penl-C6DN+siCASP2+AG490, Scale bars in FIGs. 7A-7C = 50μιη; scale bars in FIGs. 7E-7G and insets (i) and (ii) = ΙΟΟμιη.

FIGs. 8A-8I show that blocking Penl-C6DN+siCASP2-stimulated RGC axon regeneration by MAB228 and AG490 also blocks (A- J) glial activation (FIGs. 8A-8J) and reduces CNTF production in the eye (FIG. 8K). ***P<0.0001. Scale bar = 50μιη. GCL = ganglion cell layer, INL = inner nuclear layer, O L = outer nuclear layer.

FIGs. 9A-9C provide a schematic representation of a proposed mechanism of CNTF-induced gliosis in the retina after ONC and Penl-C6DN+siCASP2 treatment. After ONC, the low levels of injury-induced CNTF bind to the CNTF receptor and sub-optimally activate the JAK/STAT pathway and hence little or no axon regeneration occurs (FIG. 9A). After ONC and intravitreal delivery of Penl-C6DN+siCASP2 however, glial activation occurs in response to both injury and the presence of Penl-C6DN+siCASP2 (FIG. 9B). This gliosis leads to CNTF release, which further stimulates reactive gliosis in an autocrine manner, enhancing CNTF release. In addition, treatment with Penl-C6DN+siCASP2 also promotes release of proinflammatory cytokines from microglia that stimulate further release of CNTF from astrocytes and Muller cells. These high titres of CNTF activate the CNTF receptor and promote significant RGC axon regeneration through the JAK/STAT pathway. In contrast, blocking the gpl30 component of the CNTF receptor with MAB228 or blocking the JAK/STAT signalling pathway with AG490 blocks the autocrine response, suppresses proinflammatory cytokine-induced CNTF release leading to reduced CNTF titres, depressed reactive gliosis and hence RGC axon regeneration is prevented (FIG. 9C).

FIGs 10A-10E shows the effect of suppression of caspase-2 and caspase-6 on microglial activation in the retina. Immunohistochemistry demonstrates low levels of immunoreactive OX-42 in retinal microglia after ONC in (A) Penl controls, (B) in Penl+siCASP2 and (C) Penl-C6DN-treated retinae. Higher levels of OX-42 immunoreactivity are observed after combined Penl-C6DN+siCASP2 treatment (D), with the microglia showing an activated morphology. Quantification of the pixel intensities reflect these changes and demonstrate the significantly elevated levels of OX-42 immunoreactivity in Penl-C6DN+siCASP2-treated retinae (E). ***p<0.0001, Scale bar = 50μιη. DESCRIPTION OF SOME EMBODIMENTS OF THE INVENTION

The invention, in some embodiments thereof, relates to use of agents for inhibition of caspase-2 and caspase-6 in protection of retinal neuronal cells from apoptosis and promotion of axon regeneration.

The present inventors have shown that combined inhibition of caspase-2 and inhibition of caspase-6 activates astrocytes and Muller cells, increases ciliary neurotrophic factor (CNTF) levels in the retina and leads to enhanced RGC axon regeneration.

The present inventors delivered an inhibitor of caspase-6 (caspase-6 dominant negative (C6DN), coupled to a cell penetrating peptide, Penetratin-1 (Penl) either alone or in combination with an inhibitor of caspase-2 (chemically stabilised siRNA to caspase-2, siCASP2) and measured the effects on RGC neuroprotection and axon regeneration. It was shown that caspase-2 inhibition provided significantly more RGC neuroprotection after ONC than caspase-6 inhibition. Moreover, the combination of C6DN and siCASP2 did not potentiate RGC survival over that observed with siCASP2 alone. However, when both caspases were simultaneously inhibited, RGC axon regeneration was indirectly and significantly increased through activation of retinal glial-dependent CNTF secretion.

In dissociated adult rat mixed retinal cultures, C6DN+siCASP2 treatment also significantly increased GFAP⁺ glial activation, increased the expression of CNTF in culture, and subsequently increased the number of RGC with neurites and the mean RGC neurite length. These effects were abrogated by the addition of MAB228 (a monoclonal antibody targeted to the gpl30 component of the CNTF receptor) and AG490 (an inhibitor of the JAK/STAT pathway downstream of CNTF signalling). Similarly, in the ON crush injury model, MAB228 and AG490 neutralised C6DN+siCASP2-mediated RGC axon regeneration, Muller cell activation and CNTF production in the retina without affecting RGC survival.

It is therefore concluded that axon regeneration promoted by suppression of caspase-2 and caspase-6 is CNTF-dependent and mediated through the JAK/STAT signalling pathway. These findings offer insights for the development of effective therapeutics for promoting RGC survival and axon regeneration.

The principles, uses and implementations of the teachings herein may be better understood with reference to the accompanying description and figures. Upon perusal of the description and figures present herein, one skilled in the art is able to implement the invention without undue effort or experimentation. Before explaining at least one embodiment in detail, it is to be understood that the invention is not necessarily limited in its application to the details of construction and the arrangement of the components and/or methods set forth herein. The invention is capable of other embodiments or of being practiced or carried out in various ways. The phraseology and terminology employed herein are for descriptive purpose and should not be regarded as limiting.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the scope of the appended claims.

Citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the invention.

Experimental

Example 1: Combined suppression of caspase-2 and caspase 6 protects retinal ganglion cells from apopotosis and promoted axon regeneration

Materials and methods

Optic nerve crush (ONC)

All animal procedures were licensed and approved by the UK Home Office and the University of Birmingham Ethical Review Committee. Adult, female 6-8 week-old Sprague- Dawley rats (180-220g) were anaesthetised with Isofluorane inhalation anaesthesia (Janssen Pharmaceuticals, Oxford, UK), the ON was exposed through a supra-orbital approach and crushed bilaterally 2 mm from the globe using watchmaker's forceps as previously described by the present inventors (Vigneswara et. al, 2012). None of the animals developed cataracts, confirming that the lens had not been injured during surgery. Penl-C6DN

The mutant Caspase-6 (Cysl63Ala) dominant negative (C6DN) expression construct was a kind gift of G. S. Salvesen, Sanford-Burnham Institute, La Jolla, CA. C6DN was purified in as described by Denault and Salvensen, 2003. Penetratin-1 (Pen-1) was custom synthesized by Polypeptide Laboratories, Torrance, CA. Pen-1 and C6DN were linked by incubating equimolar amounts at 37°C for 24 hours to generate disulfide bonds. Linkage was confirmed by non-reducing 20% PAGE with western blotting using anti-His antibodies.

siCASP2 dsNA to CASP2 (siCASP2, also identified as QPI-1007) was provided by Quark Pharmaceuticals Inc., Nes Ziona, Israel. siCASP2 (QPI-1007) is a double- stranded nucleic acid compound having the structure:

5' iB - GCCAGAAUGUGGAACUCCU 3' (sense strand)

3' CGGUCUUACACCUUGAGGA 5' (antisense strand) wherein each A, C, U, and G is a ribonucleotide and each consecutive ribonucleotide is joined to the next ribonucleotide by a phosphodiester bond; wherein the sense strand comprises, counting from the 5' terminus, an unmodified ribonucleotide at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 19, a L-deoxycytidine at position 18, and an inverted deoxyabasic moiety (iB) 5' cap; and wherein the antisense strand comprises, counting from the 5' terminus, a 2'-0-Methyl sugar modified ribonucleotide at positions 2, 4, 6, 8, 11, 13, 15, 17 and 19 and an unmodified ribonucleotide at positions 1, 3, 5, 7, 9, 10, 12, 14, 16 and 18.

In vivo experiments

In the preliminary Penl-C6DN dose-finding experiment, groups comprised six rats/treatment (i.e. 12 eyes/treatment): (1) Intact, (2) ONC+Penl (vehicle control) (Penl, ΟμΜ C6DN), (3) 2μΜ Penl-C6DN, (4), 4μΜ Penl-C6DN, (5), 5μΜ Penl-C6DN and (6) 7μΜ Penl-C6DN. To monitor caspase-2 and caspase-6 activation over the first 7 days, groups of six rats/treatment (12 eyes/treatment) were killed at 4 and 7 days after ONC, while a further three rats (6 eyes) were used as intact controls. In further experiments, groups of six rats/treatment (12 eyes/treatment) were used to determine the effects of siCASP2 and pre-optimised Penl- C6DN on the levels of caspase-2 and cleaved Lamin A/C, a substrate for active caspase-6 and comprised: (1) ONC+Penl, (2) ONC+Penl-C6DN, (3) ONC+Penl+siCASP2 and (4) ONC+Penl-C6DN+siCASP2. To determine the effects of siCASP2 and pre-optimised Penl- C6DN singly and in combination on RGC survival, groups of six rats/treatment (12 eyes/treatment) were used and groups comprised: (1) ONC+PBS, (2) ONC+Penl, (3) ONC+Penl -C6DN, (4) ONC+Penl +siCASP2 and (4) ONC+Penl -C6DN+siCASP2 and (5) Intact controls. To determine the effects of siCASP2 and pre-optimised Penl-C6DN on RGC axon regeneration and Muller cell activation in the retina six rats/treatment (12 eyes/treatment) were used and groups comprised: (1) ONC+Penl, (2) ONC+Penl -C6DN, (3) ONC+Penl +siCASP2 and (4) ONC+Penl-C6DN+siCASP2. To determine the levels of CNTF in the eye, groups of six rats/treatment were used and groups comprised: (1) ONC+Penl, (2) ONC+Penl -C6DN, (3) ONC+Penl +siCASP2 and (4) ONC+Penl - C6DN+siCASP2. To determine the effects of MAB228 and AG490 on RGC survival, axon regeneration, retinal Muller cell activation and CNTF localisation, groups of twelve rats/treatment (6 rats for FG labelling and 6 rats for GAP-43 and GFAP/CNTF immunohistochemistry) were used and groups comprised: (1) ONC+Penl- C6DN+siCASP2+IgG, (2) ONC+Penl -C6DN+siCASP2+MAB228 and (3) ONC+Penl- C6DN+siCASP2+ AG490. Finally, to determine the levels of CNTF in the eye after treatment with MAB228 and AG490, groups of six rats/treatment were used and groups comprised: (1) ONC+Penl -C6DN+siCASP2+IgG, (2) ONC+Penl-

C6DN+siCASP2+MAB228 and (3) ONC+Penl-C6DN+siCASP2+AG490.

Intravitreal injections

In a preliminary experiment, Penl-C6DN was titrated to determine the optimal dose required to promote maximal RGC survival. Immediately after ONC, animals (n = 12 eyes/group) received Penl vehicle or increasing concentrations of C6DN from 2, 4, 5 and 7μΜ Penl- C6DN using glass micropipettes. In further experiments 5μΜ of Penl or Penl-C6DN was injected. Intravitreal injections were repeated every 7 days based on previous experiments by the present inventors with other caspase inhibitors (Ahmed et al, 2011). 20μg/eye of siCASP2 was injected along with either 5μΜ Penl or Penl-C6DN. The optimal dose of MAB228 was pre-determined by its ability to reduce CNTF levels in treated eyes, with optimal concentrations determined as 5μg/eye (not shown), the inhibitor of Janus-kinase 2 (JAK) AG490 was injected at 17mM/eye (Muller et al, 2007) and CNTF was injected at a dose of l ^g/eye (Muller et al, 2007). All intravitreal injections were made up in a final volume of 5μl/eye and repeated every 7 days.

Retinal wholemounts

At 19 days after ONC, 2μ1 of 4% FluoroGold (FG; Cambridge Bioscience, Cambridge, UK) in PBS was injected into the ON, between the lamina cribrosa and the site of ONC in all experimental groups (Vigneswara et al, 2012). Animals were killed 48h later and intracardially perfused with 4% formaldehyde (TAAB Laboratories, Aldermaston, UK) in PBS. Retinae were harvested and flat mounted onto Superfrost Plus microscope slides (VWR International, Lutterworth, UK). Retinal wholemounts were dried onto glass slides and mounted in Vectamount (Vector Laboratories, Peterborough, UK). Samples were then randomised by a second investigator and photographs were captured using a Zeiss Axioplan 2 fluorescent microscope equipped with a digital Axiocam FIRc camera, controlled through Axiovision 4 software (all from Zeiss, Hertfordshire, UK). The number of FG-labelled RGC was counted using the automated particle counting software in ImagePro (Version 6.0) (Media Cybernetics, Bethesda, USA) from photographs of 12 rectangular (0.36 X 0.24mm) areas/retina, 3 from each retinal quadrant, placed radially at inner (1/6 eccentricity), mid- periphery (1/2 eccentricity) and outer retina (5/6 eccentricity) from the centre of the optic disc. The number of FG-labelled RGC in the 12 images/retina were divided by the counting area and resultant numbers were pooled to calculate mean densities of FG-labelled RGC/mm² for each retina (Peinado-Ramon et al., 1996, Vigneswara et al, 2012).

Anterograde labelling of regenerating RGC axons with Rhodamine B isothiocyanate

Anterograde labelling of regenerating axons using Rhodamine B isothiocyanate (RITC; Sigma, Poole, UK) was performed as described previously (Thanos et al, 1987). Briefly, 5μ1 of a 2.5% solution of RITC was intravitreally injected using glass micropippettes 2 days prior to sacrifice. Animals were killed by overdose of C0₂, perfused with 4% formaldehyde in PBS and the ON were prepared for cryo-sectioning as described below. Tissue preparation and sectioning

After intracardiac perfusion with 4% formaldehyde in PBS, eyes and ON were removed and prepared as described previously (Douglas et al, 2009, Ahmed et al, 2010, Vigneswara et al, 2012). Briefly, eyes and ON were post-fixed in 4% formaldehyde (TAAB) in PBS, incubated in a graded series of sucrose solutions in PBS and then embedded in OCT mounting medium (Raymond A Lamb Ltd) prior to freezing at -80°C. Later, 15μπι thick parasaggital and longitudinal sections of eye and ON, respectively, were cut on a cryostat (Bright Instruments, Huntingdon, UK), adhered onto glass slides and stored at -20°C until required.

Immunohistochemistry

Immunohistochemistry was performed on sections of retina and ON as described previously (Douglas et al, 2009, Ahmed et al, 2010, Ahmed et al, 2011, Vigneswara et al, 2012). Briefly, sections were washed in PBS and non-specific binding was blocked for 20 min before incubation with the relevant primary antibody. Monoclonal anti-GAP43 (1 :500 dilution in PBS containing 3% BSA and 0.05% Tween-20; Invitrogen, Paisley, UK) was used to localise regenerating axons; monoclonal anti-GFAP and a polyclonal anti rat-CNTF (1 :500; Promega, Southampton, UK) was used to stain for astrocytes and CNTF in retinal sections and in retinal cultures; monoclonal anti-pill-tubulin (1 :200; Sigma, Poole, UK) was used to stain for RGC and their neurites in retinal cultures. Sections were washed in PBS and incubated with appropriate Alexa Fluor 488 and Texas Red-labelled secondary antibody (Invitrogen) for 1 hour at room temperature, washed, mounted in Vectashield mounting medium with DAPI (Vector Laboratories) and examined under an Axioplan-2 epi-fluorescent microscope (Zeiss).

Quantification of Miiller cell processes

Retinal Miiller cell activation was quantified as described previously (Ahmed et al, 2010). Briefly, after GFAP immunohistochemistry, GFAP⁺ Miiller cell processes were counted along a 250μπι horizontal line in retinal sections, placed orthogonal to the radial plane through the middle of the internal plexiform layer. The mean GFAP⁺ cell counts for each condition (n = 12 retinal sections/condition) were calculated and expressed as mean ±SEM. Adult rat retinal cultures

Five days after ONC and intravitreal treatments of pre-optimised reagents (concentrations detailed above), animals (n = 6 eyes/group) were killed by C0₂ overdose, retinae harvested and dissociated using a Papain dissociation system following the manufacturer's instructions (Worthington Biochemical, Lakewood, NJ, USA), as described previously (Vigneswara et al, 2013). Retinal cells from untreated rats were also prepared in a similar way. Retinal cells were plated at a density of 125 x 10³/well into 8-well chamber slides pre-coated with laminin and poly-D-lysine and cultured in 300μ1 Neurobasal-A medium supplemented with B27 supplement and gentimicin (all from Invitrogen, Paisley, UK). Cells were cultured for 4 days at 37°C and 5% C0₂ before either removal of culture medium and cells for ELISA (see below for description) or fixation in 4% formaldehyde in PBS for immunocytochemistry. Experiments comprised of n = 3 wells/treatment and each experiment was repeated on 3 separate occasions and hence results for each data point are the mean ±SEM from 9 wells/treatment.

Immunocytochemistry

Fixed cells were immunostained for GFAP and βΙΙΙ-tubulin to determine the number of GFAP⁺ astrocytes and Muller cells and to measure the number of RGC with neurites and the neurite lengths, as described previously (Ahmed et al, 2010). Briefly, cells were washed in PBS, permeabilised and blocked using PBS containing 3% BSA and 0.1% Triton X-100 for 30 min at room temperature before incubation with the mouse anti-GFAP (1 :500 dilution, Sigma, Poole, UK) or mouse anti-pill-tubulin antibodies (1 :200 dilution, Sigma) for 1 hour at room temperature in a humidified chamber. Cells were then washed in PBS and incubated for 1 hour at room temperature with Alexa-488 anti-mouse IgG (1 :400 dilution; Invitrogen). After further washes in PBS, sections were mounted under coverslips using Vectamount containing DAPI (Vector Laboratories, Peterborough, UK) and viewed under a Zeiss Axioplan 2 fluorescent microscope equipped with an Axiocam URc and Axiovision software, as described earlier. Immunocytochemistry controls with primary antibody omitted were included in each run and the negative control slides were used to set the background threshold levels for non-specific staining (not shown) during image capture. RGC survival and neurite outgrowth and quantification of astrocyte activation

The mean number of surviving pill-tubulin⁺ RGC, mean neurite length, the number of RGC with neurites and the number of GFAP⁺ astrocytes/Muller cells were quantified as described previously (Vigneswara et al, 2013). Briefly, each anonymised chamber slide was divided into 9 quadrants and images of RGC, their neurites and GFAP⁺ astrocytes/Muller cells were captured randomly from each quadrant. Axiovision (Zeiss) was then used to measure the neurite lengths, while ImagePro (Version 6.3; Media Cybernetics, Bethesda, MD, USA) was used to quantify the number of pill-tubulin⁺ RGC with neurites longer than the RGC diameter and the number of GFAP⁺ astrocytes/Muller cells. Neurite outgrowth from at least 180 RGC/treatment was measured, except in untreated and Pen 1 -vehicle treated cultures in which 100 RGC were assessed (i.e. all RGC that grew neurites longer than the RGC diameter).

ELISA for CNTF

For detection of CNTF in vitro, cultured cells and culture medium were homogenised in cell lysis buffer and clarified by centrifugation. Lysates were then assayed for CNTF and compared to culture medium only conditions to account for any background CNTF. Retinae harvested from in vivo experiments were homogenised in cell lysis buffer, clarified by centrifugation and the supernatant was frozen at -20°C until required for assay. A commercially available rat CNTF ELISA kit (R&D Systems, Oxford, UK) was used to detect CNTF in cultured retinal cell lysates, following the manufacturer's instructions.

Protein extraction and Western blotting

Six rats (12 eyes/treatment) were killed by an overdose of C0₂ and total protein from retinae and where appropriate, ON, were extracted in cell lysis buffer and processed for Western blotting as previously described (Ahmed et al., 2005, Ahmed et al, 2006, Ahmed et al, 2010). Western blots were probed overnight at 4°C with antibodies against: rabbit anti-human caspase-2 (CASP2; Abeam); rabbit anti-Lamin A/C and rabbit anti-human GAPDH, all from Cell Signalling Technology (Danvers, MA, USA). Relevant protein bands were detected with an appropriate HRP-labelled secondary antibody (GE Healthcare, Buckinghamshire, UK) and detected using an enhanced chemiluminescence system (ECL) (GE Healthcare). Blots were stripped and re-probed as required. Densitometry

Western blots were quantified by densitometry as described previously (Ahmed et al, 2006, Douglas et a/., 2009, Ahmed et a/., 2010). Briefly, blots were scanned into Adobe Photoshop and TIFF files were analysed in Scionlmage (version 4.0.2, Scion Corp, Maryland, USA) using the built-in gel plotting macros. The integrated density of each band of interest in each lane was calculated for 3 separate blots from 3 independent experiments.

Statistical analysis

The significance of differences between sample means were calculated using GraphPad Prism (GraphPad Software Inc., Version 4.0, CA, San Diego, USA) by one-way analysis of variance (ANOVA) followed by post-hoc testing with Dunnett's method.

Results

Characterization of a specific inhibitor of caspase-6

To determine the functional relevance of the up-regulation of cleaved caspase-6 (C-CASP6) expression after ONC, caspase-6 activity was inhibited in the retina. A previously characterized C6DN construct (Edgington et a/., 2012) was used to block activation/activity of caspase-6. For access into the retina, C6DN was disulphide-linked to Penetratin-1 (Penl), a cell penetrating peptide (Davidson et a/., 2004), the disulphide bond was broken by the reducing environment of the cell cytoplasm, thus releasing the peptide cargo and allowing it to act at its cellular target.

The neuroprotective efficacy of this construct was examined by assessing RGC survival after ONC and intravitreal delivery of a range of doses of Penl-C6DN, by counting the number of FluoroGold (FG) backlabelled RGC in retinal wholemounts. Compared to the vehicle treatment (Penl), which left 400 ± 45 FG-labelled RGC/mm², Penl-C6DN (2, 4 and 5μΜ) caused a dose-dependent statistically significant increase in the numbers of surviving FG- labelled RGC, to a maximal level of 987 ± 65 RGC/mm² (Fig. 1A-F). Concentrations of Penl-C6DN above 5μΜ did not significantly increase the number of FG-labelled RGC; thus, maximal RGC protection (60%) was observed with 5μΜ Penl-C6DN. These results demonstrated that specific blockade of caspase-6 activity, using Penl-C6DN, significantly enhances RGC survival. To ensure that each intervention affected only the targeted caspase, the expression of caspase- 2 was examined in retinae treated with Penl and Penl-C6DN by western blot. No decrease in caspase-2 levels was detected (Figs. 2A and 2B). Penl-C6DN decreased basal cleaved Lamin A/C (a substrate of caspase-6 (Orth et al, 1996, Takahashi et al., 1996, Ruchaud et al, 2002, Mintzer et al., 2012)) levels while, in Penl and Penl+siCASP2-treated retinae, basal cleaved Lamin A/C levels were unaffected (Fig. 2A and 2B), indicating constitutive caspase-6 activity in non-RGC cells. These results demonstrated that the caspase-2 and caspase-6- specific inhibitors disclosed herein do not have cross reactivity but that each specifically regulates their targeted caspase.

To assess the neuroprotective properties of the combined Penl-C6DN and siCASP2, optimal doses of Penl-C6DN were intravitreally injected together with previously optimised dose of siCASP2 (Ahmed et al, 2011) after ONC. The number of FluoroGold backfilled surviving RGC were assessed at 21 days after ONC. In eyes treated with the vehicle control (Penl), 405 ± 34 RGC/mm² remained at 21 days after ONC (Figs. 2C and 2D), while in Penl-C6DN treated eyes 979 ± 34 RGC/mm² remained (Figs. 2C and 2D). However, Penl+siCASP2 protected 2145 ± 54 RGC/mm², while Penl-C6DN+siCASP2 did not significantly improve RGC neuroprotection over that observed for siCASP2 alone (Figs. 2C and 2D). Compared to intact controls, Penl-C6DN promoted 60% RGC neuroprotection while siCASP2 alone or Penl-C6DN+siCASP2 protected >95% of RGC from apoptosis at 21 days after ONC. These results suggest that both Penl+siCASP2 and Penl-C6DN+siCASP2 promote optimal RGC survival.

Penl-C6DN+siCASP2 promoted RGC axon regeneration

Previous studies have suggested that active caspase-2 and caspase-6 modulate different signalling pathways. The present inventors therefore tested whether simultaneous blocking of these caspases using a combined treatment of Penl-C6DN and siCASP2 would promote RGC axonal regeneration. It has been previously shown that siCASP2 decreased caspase-2 expression by almost 90% after ONC but there was little associated RGC axonal regeneration (Ahmed et al, 201 1). In other models of axonal degeneration (trophic factor deprivation and stroke) caspase-6 activation initiates axonal degeneration (Nikolaev et al, 2009, Akpan et al, 2011). To determine whether caspase-6 might have a similar function in RGC degeneration, caspase-6 function was inhibited using C6DN and combined with delivery of siCASP2 to promote optimal RGC survival and thus enhance the possibility of reduced RGC loss and axon degeneration. At 7 and 14 days after ONC, animals received intravitreal injections of siCASP2 and/or Penl-C6DN, and analysed RGC axon regeneration was analyzed through the crush site at 21 d after ONC using antibodies against GAP-43, a marker for regenerating axons (Berry et al, 1996, Leon et al, 2000).

When the ON of Penl treated animals were sectioned longitudinally and immunostained for GAP-43 (Figs. 3 A and 3E), few GAP43⁺ axons were seen in the proximal ON segment, and none traversed the lesion site to enter the distal ON segment. In Penl-C6DN treated ON (Figs. 3B and 3E), more GAP43⁺ axons occupied the proximal ON but few traversed the lesion site, although some of these penetrated into the distal ON up to 1500μιη from the lesion centre. The mean number of axons/ON were low after the monotherapies, so that the numbers of GAP43⁺ axons were similar in the Penl+siCASP2-treated and Penl-C6DN- treated groups, with only occasional axons penetrating >1000μιη from the lesion centre and into the distal ON (Figs. 3C and 3E). The greatest numbers of GAP43⁺ axons were present in the Penl-C6DN+siCASP2-treated groups, with 195 ± 9 and 72 ± 8 axons/section growing at ΙΟΟΟμπι and 2500μιη, respectively, from the lesion (Figs. 3D and 3E). These results suggest that, while Penl-C6DN or siCASP2 alone promote little RGC axon regeneration, combined delivery of Penl-C6DN+siCASP2 promotes significant RGC axon regeneration.

Anterograde labelling with Rhodamine B isothiocyanate confirmed similar numbers of regenerating RGC axons emerging from the lesion site and growing through the distal optic nerve stump (arrowheads) to 1000 and 2000μπι from the lesion site (Fig 3F-H). For example, the number of RITC-labelled axons at 250, 1000 and 2000μπι from the lesion site were, 200 ± 14, 210 ± 12 and 30 ± 10 axons/section, respectively (not illustrated).

Penl-C6DN+siCASP2 delivery enhanced retinal glia activation and upregulated CNTF production in glia and occasional RGC

Studies of retinal glial activation in models of RGC axon regeneration have shown that glial activation is correlated with axon regeneration (Berry et al, 1996, Leon et al, 2000, Lorber et al, 2002, Yin et al, 2003, Lorber et al, 2005, Pernet and Di Polo, 2006, Muller et al, 2007, Lorber et al, 2008, Lorber et al, 2009, Ahmed et al, 2010, Lorber et al, 2012). It has also been shown that after ONC, inflammation-mediated glial activation in either the vitreous or in the ON injury site promoted significant RGC survival, but that only vitreal inflammation with associated retinal glial activation was correlated with axon regeneration. This observation suggests that activated retinal glia secrete factors that are conducive for RGC axon regeneration (Ahmed et al, 2010). Moreover, activated retinal astrocytes express CNTF in response to lens injury or intravitreal Zymosan injections; the downstream JAK/STAT3 pathway is strongly activated in regenerating RGC and the lens injury-induced switch of RGC to a regenerative state is dependent on CNTF and JAK/STAT3 signalling (Muller et al, 2007). Thus, it was investigated whether intravitreal delivery of Penl- C6DN+siCASP2 after ONC also promoted retinal glial activation and CNTF expression.

GFAP⁺ astrocytes/Miiller cell end-feet were similarly activated and low levels of CNTF appeared in the nerve fibre layer (NFL) after ONC in vehicle control Penl-treated (Figs. 4A- 4C) and in Penl-C6DN-treated eyes (Figs. 4D-4E). In Penl+siCASP2-treated eyes (Figs. 4G- 41), more GFAP⁺ astrocytes/Miiller cells and CNTF were observed in the NFL compared to Penl and Penl-C6DN treated eyes. The greatest levels of GFAP⁺ and CNTF⁺ staining were observed in Penl-C6DN+siCASP2-treated eyes, with more numerous GFAP Muller cell processes spanning the entire radial width of the retina (Figs. 4J-4L), reflected by twice the number of activated Miiller cell processes counted in these eyes compared to the numbers in other treatments (Fig. 4M). In addition, occasional RGC were immunopositive for CNTF only after combined suppression of caspase-2 and -6, suggesting that some RGC may also respond to the effects of the combined treatments (Figs. 4N and 40).

ELISA confirmed higher titres of CNTF in the retina of eyes treated with Penl- C6DN+siCASP2 than that measured in the eyes from any other treatments (Fig. 4P). Together, these results suggest that the bi-therapy of Penl-C6DN+siCASP2 activates retinal astrocytes/Miiller cells to produce high titres of CNTF and that this may explain the enhanced RGC axon regeneration observed after this combined treatment.

Another possible mechanism of the enhanced CNTF production by retinal glia and RGC was investigated, since caspase-6 suppression can up-regulate proinflammatory cytokine release by microglia that may then facilitate enhanced CNTF release from activated retinal cells. Immunohistochemistry for OX-42, a marker of microglia, demonstrated higher levels of microglial activation after caspase-2 and caspase-6 monotherapy suppression, and that combined suppression of caspase-2 and caspase-6 synergistically increased this microglial activation (Figs. 10A-10E). These results suggest that microglial activation and subsequent proinflammatory cytokine production may contribute to the activation of retinal glia and subsequent CNTF release. RGC cultures prepared from ONC+Penl-C6DN+siCASP2 show enhanced glial activation and significant titres of CNTF in culture medium

Many of the responses observed in vivo after ONC and treatment may be studied in vitro using adult retinal cultures. To further assess whether Penl-C6DN+siCASP2 promoted glial activation and indirectly enhanced neurite outgrowth of surviving RGC, retinal cultures from intact untreated eyes and from eyes 5 days after ONC and treatment with Penl vehicle, Penl- C6DN, Penl+siCASP2 and Penl-C6DN+siCASP2 treatment were prepared. After 3 days in culture, there were no activated GFAP⁺ glia detected in retinal cell cultures derived from untreated eyes (Figs. 5A and 51). In retinal cell cultures prepared after ONC (Fig. 5B and I) and ONC followed by Penl treatment (Fig. 5C and I), 1396 ± 259 and 1367 ± 379 activated GFAP⁺ glia were present, respectively. Cultures prepared from eyes after ONC and monotherapy with Penl-C6DN and Penl+siCASP2, contained enhanced numbers of activated GFAP⁺ glia (2867 ± 379 and 2433 ± 208, Figs. 5D, 5E and 51), respectively, whilst cultures prepared from Penl-C6DN+siCASP2-treated eyes contained the greatest numbers of GFAP⁺ glia (4900 ± 355 glia, Figs. 5F and 51). These results demonstrated that Penl- C6DN+siCASP2 treatment led to the survival of enhanced numbers of activated GFAP⁺ glia in retinal cell cultures and this confirmed the in vivo findings.

Since reactive astrocyte gliosis can be induced by elevated levels of CNTF (Winter et al, 1995, Escartin et al., 2007, Kirsch et al, 2010) the present inventors investigated whether blockade of the gpl30 signalling component of the CNTF receptor complex using MAB228 or of the JAK/STAT3 pathway using AG490 suppressed the Penl-C6DN+siCASP2-mediated survival of activated glia in retinal cell cultures. In cultures prepared from eyes after ONC and treatment for 5 days with MAB228 and AG490, it was observed that the Penl- C6DN+siCASP2-mediated enhancement of GFAP⁺ glia numbers in retinal cultures was depressed to control ONC and ONC+Penl levels (Figs. 5G, 5H and 51, respectively) by the inhibitors of CNTF signalling.

Since high levels of CNTF were observed after ONC and treatment with Penl- C6DN+siCASP2 in vivo, ELISA was used to monitor CNTF titres in defined cell culture medium after treatment. It was demonstrated that in the media of cultures prepared from untreated, ONC- and Penl- treated retinae, low levels of CNTF were detected (Fig. 5J) but, in cultures from both Penl-C6DN and Penl+siCASP2-treated retinae, approximately 38 ± 8 ng/mg of CNTF protein were present in the media. The levels of released CNTF were 3-fold higher in cultures prepared from retinae after Penl-C6DN+siCASP2 bi-therapy (115 ± 12 ng/mg of protein). However, addition of the inhibitors of CNTF signalling, MAB228 or AG490, depressed the numbers of Penl-C6DN+siCASP2 activated glia and the released CNTF levels to those observed in the cultures from control ONC and Penl-treated eyes. These results suggested that Penl-C6DN+siCASP2 activates retinal glia and CNTF production, while blockade of CNTF signalling not only suppresses the numbers of activated glia but also CNTF production in culture.

Treatment of retinal cell cultures with Penl-C6DN+siCASP2 promotes RGC neurite outgrowth

Since Penl-C6DN+siCASP2 treatment activates retinal glia and secretion of CNTF in vivo and in vitro, it was hypothesized that RGC survival and neurite outgrowth would also be enhanced in these cultures. The present inventors therefore investigated whether Penl- C6DN+siCASP2 bi-therapy led to stimulated RGC survival and neurite outgrowth in retinal cell cultures, and whether MAB228 and AG490 treatment impacted on the RGC survival and neurite outgrowth. In retinal cultures from untreated (Figs. 6A and 6I-6K) and Penl-treated eyes (Figs. 6A and 6I-6K), very few RGC survived and grew neurites. After Penl+CNTF treatment, RGC survival was increased by 1.7-fold, while the number of RGC with neurites increased to 25 ± 5 RGC with mean neurite lengths of 100 ± ΙΟμιη recorded, compared to Penl alone (Figs. 6B and 6I-6K). The addition of Penl-C6DN (Figs. 6C, 61 and 6J) and Penl+siCASP2 on their own (Figs. 6D, 61 and 6J) promoted 50% fewer RGC with neurites (10-12) than that seen with CNTF, with RGC neurite lengths of between 45-50μιη. RGC survival was similar in Penl-C6DN and Penl+CNTF treatment groups, while Penl+siCASP2 provided greater than 2-fold more RGC survival than CNTF or Penl-C6DN (Fig. 6K). Combined Penl-C6DN+siCASP2 treatment significantly increased the number of RGC with neurites and the mean neurite length to 130 ± 8 μπι (Figs. 6E and 61) compared to CNTF (Figs. 61 and 6 J). RGC survival in the Penl-C6DN+siCASP2 and Penl+siCASP2 groups was similar, but was significantly greater than that seen with the CNTF, Penl-C6DN or control groups (Fig. 6K). Despite the observation that RGC neurite outgrowth was completely suppressed to basal levels in Penl-C6DN+siCASP2 cultures treated with MAB228 and AG490 (Figs. 6G-6J), RGC survival remained at the same high levels as other groups containing siCASP2 (Fig. 6K). ELISA to detect the levels of CNTF present in culture media from the different treatment groups showed that cultures prepared from eyes treated with Penl -CNTF contained 23 ± 3ng/ml, whilst those treated with Penl-C6DN and Penl+siCASP2 contained 20 ± 3 and 16 ± 2.5ng/ml (Fig. 6L). However, the highest levels of CNTF (64 ± 5.5ng/ml) were detected in cultures prepared from Penl-C6DN+siCASP2- treated eyes, equating to 3 -fold more CNTF than after treatment with Penl-CNTF. These results suggest that RGC survival is maximal in the presence of siCASP2, while C6DN+siCASP2-mediated glial activation and subsequent high titres of CNTF secretion enhances RGC neurite outgrowth.

Blocking gpl30 or the JAK/STAT pathway blocked Penl-C6DN+siCASP2-mediated RGC axon growth and suppressed glial activation and CNTF production in the retina

Since suppression of gpl30 and the JAK/STAT pathway blocked Penl-C6DN+siCASP2- mediated RGC neurite outgrowth in vitro without affecting RGC survival, it was considered whether MAB228 and AG490 could also attenuate Penl-C6DN+siCASP2-induced RGC axon growth in vivo and assessed the impact of these treatments on RGC survival after ONC. It was shown that after intravitreal injection of Penl-C6DN+siCASP2, RGC survival was unaffected by MAB228 or AG490 treatment and remained at the same high levels as treatment with a control IgG (Figs. 7A-7D). RGC axon regeneration in Penl- C6DN+siCASP2+IgG control treatment group was the same as that observed earlier (comparing Figs. 7E and 7H with Fig. 3). However, the Penl-C6DN+siCASP2-stimulated RGC axon regeneration was almost completely blocked by intravitreal injection of either MAB228 (Figs. 7F and 7H) or AG490 (Figs. 7G and 7H), suggesting that the CNTF pathway was primarily involved in Penl-C6DN+siCASP2-mediated RGC axon regeneration.

The present inventors then investigated whether MAB228 and AG490 also blocked Penl- C6DN+siCASP2-mediated glial activation and CNTF production in the retina. After intravitreal Penl-C6DN+siCASP2 plus control IgG injections, abundant GFAP and CNTF⁺ staining was observed in astrocytes of the NFL and Muller cell radial processes (Figs. 8A- 8C). However, in Penl-C6DN+siCASP2+MAB228 (Figs. 8D-8F) and Penl- C6DN+siCASP2+AG490-treated eyes (Figs. 8G-8I), GFAP and CNTF⁺ staining was markedly attenuated. The mean number of activated Muller glia processes in Penl- C6DN+siCASP+IgG treated retinae was 210 ± 5mm^"1 compared to only 45 ± 5 and 8 ± 3 processes/mm^"1 in Penl-C6DN+siCASP2+MAB228 and Penl-C6DN+siCASP2+AG490, respectively (Fig. 8J). ELISA to quantify the levels of CNTF in Penl-C6DN+siCASP2+IgG- treated retinae measured 223 ± 5 ng/mg of CNTF but the growth factor was barely detectable in both Penl-C6DN+siCASP2+ MAB228 and Penl-C6DN+siCASP2+AG490-treated retinae (Fig. 8K).

Taken together, these results suggest that Penl-C6DN+siCASP2-mediated RGC axon regeneration is suppressed when gpl30 and JAK/STAT are blocked, and this correlates with reduced retinal glial activation and CNTF levels. Thus, inhibition of caspase-6, while capable of promoting optimum RGC survival when combined with inhibition of caspase-2, might be a promising way of indirectly enhancing RGC axon regeneration after optic nerve trauma.

Discussion

The data presented here demonstrate that despite low levels of detectable caspase-6 activation after ONC, inhibition of caspase-6 promoted 60% RGC survival while inhibition of caspase-2 promoted greater than 95% RGC survival. Combined inhibition of caspase-6 and caspase -2 protected the majority of RGC from death but also promoted extensive retinal gliosis and significant RGC neurite outgrowth/axon regeneration through the ON lesion site and along the distal ON. RGC axon regeneration promoted by combined suppression of caspase-6 and caspase-2 was mediated by glial-derived CNTF since blockade of the CNTF receptor or inhibition of JAK/STAT signalling, suppressed the associated glial activation, blocked CNTF production, inhibited RGC axon regeneration and preserved RGC viability. Taken together, the results show that combined suppression of caspase-2 and caspase-6 is RGC neuroprotective and activates a novel indirect RGC axogenic effect mediated by caspase- dependent gliosis and subsequent release of CNTF and JAK/STAT signalling.

Combined caspase-6 and caspase-2 suppression did not induce stronger neuroprotection than that observed with caspase-2 suppression alone, since suggesting that RGC death is predominantly mediated by caspase-2 and not caspase-6.

The sub-optimal levels of RGC neuroprotection seen after C6DN treatment, suggest that use of an effective caspase-6 inhibitor may increase RGC survival by achieving greater levels of caspase-6 down-regulation/inhibition. For example, C6DN suppressed Lamin A/C cleavage, a primary target of caspase-6 activity, by only 60% compared to treatment groups that did not contain C6DN (see Fig. 2A). Greater levels of caspase-6 down-regulation may more effectively suppress Lamin A/C cleavage indicating a more effective inhibition of caspase-6 activity; more effective inhibition of caspase-6 could lead to a greater stimulation of CNTF production, and better RGC survival and axon regeneration. The involvement of caspases in axon regeneration

The data presented here demonstrate that inhibition of caspase-6 along with down-regulation of caspase-2 promoted significant RGC axon regeneration and that this regeneration was approximately 10-fold greater than that observed using a pseudopeptide caspase inhibitor in previous studies (Monnier et al, 2011). Notably, combined inhibition of caspase-2 and caspase-6 significantly activated retinal glia, including Miiller cells and astrocytes, and stimulated the production of high levels of CNTF both in vivo and in vitro. Moreover, in combined caspase-2 and caspase-6 suppressed retinal cultures and in eyes, blocking CNTF receptor function and JAK/STAT signalling both prevented glial activation, suppressed CNTF production and blocked RGC neurite/axon regeneration. These observations all suggest a link between caspase-6 and retinal glia.

Furthermore, inhibition of caspase-6 and caspase-2 promoted GFAP and CNTF expression in the glia present in the mixed retinal cultures, indicating an indirect mechanism of enhancing RGC survival and axon regeneration. Thus, without wishing to be bound by theory, the combination of caspase-2 and caspase-6 stimulates retinal gliosis, which in turn upregulates the expression CNTF, and probably other neurotrophic factors, that then promote RGC survival and axon regeneration.

Furthermore, without being bound by theory, the current study implies differential mechanisms for RGC survival and axon regeneration and suggests that retinal glia are important to both processes. For example, the present inventors suggest that the activation of retinal glia that occurs after ONC is enhanced by Penl-C6DN+siCASP2, increasing the release of CNTF. CNTF then acts not only on RGC in a paracrine manner to increase their regenerative capacity but also induces by an autocrine mechanism further reactive gliosis in astrocytes and Miiller cells (DeChiara et al, 1995, Kahn et al, 1995, Winter et al, 1995, Escartin et al, 2006, Escartin et al, 2007), which in turn enhances CNTF production, contributing to an additional regenerative ability of RGC (Figure 9). CNTF is probably also released by RGC through autocrine mechanisms (Figure 9) contributing to the higher titres of CNTF in the combined Penl-C6DN+siCASP2-treated retinae. This proposed mechanism might explain why enhanced RGC axon regeneration occurs after Penl-C6DN+siCASP2 treatment. In contrast, blocking gpl30 or the JAK/STAT pathway does not prevent the low levels of CNTF production by injury-induced gliosis but appears to suppress the C6DN- induced gliosis and subsequent high titre release of CNTF, which in turn reduces the regenerative response of RGC. Mechanisms of caspase-6-modulated retinal gliosis and CNTF expression

The present inventors observed that suppression of caspase-6 activates retinal microglia (Figure 10A-10E) and thus may contribute to the release of pro-inflammatory cytokines. Receptor binding of proinflammatory cytokines released from activated astrocytes and microglia activates a variety of intracellular signaling pathways, including the c-Jun N- terminal kinase (INK), p38 mitogen activated protein kinase (p38/MAPK), PI3 kinase, extracellular signaling-related kinase (ERK) and activation of caspase-1 and -3 (Van Eldik et al, 2007, Anisman, 2009). Activation of J K and p38/MAPK is known to contribute to the accumulation of GFAP in astrocytes (Tang et al, 2006) and thus release from retinal glia of gpl30 receptor ligands (LIF and IL-6) which conspire with CNTF to signal RGC survival through the JAK/STAT pathway. Indeed, CNTF is normally released by astrocytes and Miiller cells of the retina after ONC, probably mediated by the release of inflammation- induced cytokines such as interleukin-ΐβ and TNF-a, all of which enhance CNTF release (Kamiguchi et al, 1995, Muller et al, 2007, Lorber et al, 2008, Lorber et al, 2012). CNTF induces changes in astrocyte responses including upregulation of GFAP, cellular hypertrophy and metabolic changes, that lead to the expression of further CNTF.

In conclusion, the present findings demonstrate that bi-therapies that suppress caspase-2 and caspase-6 inhibit RGC apoptosis and promote retinal gliosis, leading to the release of high titres of CNTF, which promotes RGC axon regeneration. These studies suggest that the collective targeting of caspase-2 and caspase-6 has therapeutic potential in treating human adult nervous system trauma and disease.

Example 2: Animal Models for Testing dsRNA Compounds in Spinal Cord Injury

In a non-limiting example, testing of the compositions disclosed herein comprising dsRNA inhibitors for treating spinal cord injury is performed in the rat spinal cord contusion model as described by Young, 2002 (Young, 2002). Other predictive animal models of spinal cord injury are described in the following references: Gruner JA, 1992; Hasegawa, 2003; Huang and Young, 1994 (Gruner, 1992, Huang and Young, 1994, Hasegawa and Grumet, 2003). Pharmaceutical compositions comprising a caspase-2 inhibitor and a caspase-6 inhibitor are tested in these animal models, which shows that these compositions treat spinal cord injury. Example 3: Rat Models for Testing the caspase-2 inhibitor and the caspase-6 inhibitor in CNS Injury

Closed Head Injury (CHI): Experimental traumatic brain injury (TBI) produces a series of events contributing to neurological and neurometabolic cascades, which are related to the degree and extent of behavioral deficits. CHI is induced under anesthesia, while a weight is allowed to free-fall from a prefixed height (Chen et al, 1996) over the exposed skull covering the left hemisphere in the midcoronal plane.

Transient middle cerebral artery occlusion (MCAO): A 90 to 120 minutes transient focal ischemia is performed in adult, male Sprague Dawley rats, 300-370 gr. The method employed is the intraluminal suture MCAO (Longa et al, 1989, Dogan et al, 1999). Briefly, under halothane anesthesia, a 3-0-nylon suture material coated with Poly-L-Lysine is inserted into the right internal carotid artery (ICA) through a hole in the external carotid artery. The nylon thread is pushed into the ICA to the right MCA origin (20-23 mm). 90-120 minutes later the thread is pulled off, the animal is closed and allowed to recover.

Permanent middle cerebral artery occlusion (MCAO): Occlusion is permanent, unilaterally- induced by electrocoagulation of MCA. Both methods lead to focal brain ischemia of the ipsilateral side of the brain cortex leaving the contralateral side intact (control). The left MCA is exposed via a temporal craniotomy, as described for rats (Tamura et al, 1981). The MCA and its 20 lenticulo striatal branch are occluded proximally to the medial border of the olfactory tract with microbipolar coagulation. The wound is sutured, and animals returned to their home cage in a room warmed at 26°C to 28°C. The temperature of the animals is maintained all the time with an automatic thermostat. Evaluation Process: The efficacy of the pharmaceutical compositions disclosed herein for treating CNS injury is determined by mortality rate, weight gain, infarct volume, short and long term clinical, neurophysiological and behavioral (including feeding behavior) outcomes in surviving animals. Infarct volumes are assessed histologically (Mintorovitch et al, 1991, Knight et al, 1994). The staircase test (Montoya et al, 1991) or the motor disability scale according to Bederson's method (Bederson et al, 1986) is employed to evaluate the functional outcome following MCAO. The animals are followed for different time points, the longest one being two months. At each time point (24 hours, 1 week, 3, 6, 8 weeks), animals are sacrificed and cardiac perfusion with 4% formaldehyde in PBS is performed. Brains are removed and serial coronal 200 μπι sections are prepared for processing and paraffin embedding. The sections are stained with suitable dyes such as TCC. The infarct area is measured in these sections using a computerized image analyzer.

Pharmaceutical compositions comprising a RTP801 inhibitor and a Casp2 inhibitor are tested in this animal model, which shows that these compositions treat and/or prevent CNS injury.

Example 4: APP Transgenic Mouse Model of Alzheimer's Disease

Animals and Treatment: The study includes twenty-four (24) APPV717I transgenic mice (female), a model for Alzheimer's disease (Moechars et al., 1996, Moechars et al., 1999), aged 11 months that are randomly divided into two equal groups (Group I and Group II). Animals are treated with a pharmaceutical composition comprising at least one siRNA compound directed at the RTP801 and/or Casp2 gene. Animals in control groups are treated with a vehicle solution. Compositions comprising the following concentrations of siRNA are tested: (i) 100 μg of siRNA compound/ 3 μΐ of vehicle; (ii) 200 μg of siRNA compound/3 μ 1 of vehicle and (iii) 500 μg of siRNA compound / 3 μΐ of vehicle. Compositions comprising the following vehicle are tested: (i) 5% glycerol solution; (ii) 10% glycerol solution and (iii) 15% glycerol solution. In this study the compositions are administered once every 4 days, during 3-4 month period of the experiment.

Termination: Mice are sacrificed; brains are dissected and processed as follows: one hemisphere for histological analysis and one hemisphere for molecular biology analysis.

Evaluation Process: The following histological analysis is performed:

1. Anti-amyloid β (Αβ) staining and quantification (4 slides/mouse)

2. Thioflavin S staining and quantification of Αβ plaques(4 slides/mouse)

3. CD45 staining and quantification (4 slides/mouse)

4. GFAP (astrocytosis) staining and quantification

Results: Pharmaceutical compositions comprising a caspase-2 inhibitor and a caspase-6 inhibitor are tested in this animal model, which shows that these composition are useful in treating Alzheimer's disease. Example 5: Mouse Model of ALS

Objective: To examine the efficacy of a caspase-2 inhibitor and a caspase-6 inhibitor in the mutant S0D1^G93A mouse model of ALS.

Animals and Treatment: Animals in test group are treated with at least one inhibitor of the caspase-2 gene and at least one inhibitor of the caspase-6 gene. Animals in control group are treated with a composition comprising a control compound. Animals in vehicle group are treated with a vehicle solution. In this study the compositions are administered once every 4 days, starting from 30 days of age.

Analysis of disease progression: Behavioral and electromyography (EMG) analysis in treated and untreated mice is performed to monitor disease onset and progression. Mice are pretested before start of treatment, followed by weekly assessments. All results are compared statistically. The following tests are performed:

1. Swimming tank test: this test is particularly sensitive at detecting changes in hind-limb motor function (Raoul et al., 2005).

2. Electromyography: EMG assessments are performed in the gastrocnemius muscle of the hind limbs, where compound muscle action potential (CMAP) is recorded (Raoul et al, 2005).

3. Body weight: The body weight of mice is recorded weekly, as there is a significant reduction in the body weight of SOD1G93A mice during disease progression (Kieran et al, 2007). Assessment of lifespan: The lifespan in days for treated and untreated mice is recorded and compared statistically to determine whether treatment by administering a caspase-2 inhibitor and a caspase-6 inhibitor implicated in ALS has any significant effect on lifespan. Mice are sacrificed at a welldefined disease end point, when they have lost >20% of body weight and are unable to raise themselves in under 20 seconds. All results are compared statistically.

Post mortem histopathology: At the disease end-point mice are terminally anaesthetized and spinal cord and hind-limb muscle tissue are collected for histological and biochemical analysis.

Examining motor neuron survival: Transverse sections of lumbar spinal cord are cut using a cryostat and stained with gallocyanin, a nissl stain. From these sections the number of motor neurons in the lumbar spinal cord is counted (Kieran et al, 2007), to determine whether dsRNA treatment prevents motor neuron degeneration in S0D1^G93A mice.

Examining spinal cord histopathology: Motor neuron degeneration in S0D1^G93A mice results in astrogliosis and activation of microglial cells. Here, using transverse sections of lumbar spinal cord the activation of astrocytes and microglial cells is examined using immunocytochemistry to determine whether combined caspase-2 inhibitor and caspase-6 inhibitor treatment reduced or prevented their activation.

Examining muscle histology. Hind-limb muscle denervation and atrophy occur as a consequence of motor neuron degeneration in S0D1^G93A mice. At the disease end point the weight of individual hind-limb muscles (gastrocnemius, soleus, tibialis anterior, extensor digitorium longus muscles) is recorded and compared between treated and untreated mice. Muscles are then processed histologically to examine motor end plate denervation and muscle atrophy (Kieran et al, 2005).

Example 6: Model Systems of Huntington's Disease (HP) in Mice

In a non-limiting example, testing of the compositions disclosed herein for treating Huntington's disease is performed in the HD mouse model, R6/2 (Wang et al, 2005).

Results: The compositions and methods disclosed herein are tested in this animal model, and show efficacy in treating Huntington's disease.

Example 7: Evaluation of Protection of Retinal Ganglion Cell Dendrites after Axonal Injury in RGC-YFP Transgenic Mice Model

Experimental animals and surgical procedure: Experimental procedures are carried out on C57BL/6 transgenic or wild-type control mice. Adult transgenic mice carrying the yellow fluorescent protein (YFP) gene under control of the Thy-1 promoter (YFP-H line, Jackson Laboratory, Bar Harbor, ME, USA;) are studied (Feng et al, 2000). Approximately 10-30% of retinal ganglion cells are exclusively labelled in the retina of these transgenic mice. All surgical procedures were carried out on 3 to 7 month-old mice under general anesthesia (2% Isoflurane; 0.8 L/min). Optic nerve axotomy: The optic nerve axotomy is carried out on mice as previously described (Lebrun-Julien et al, 2009). Briefly, the left optic nerve iss exposed and carefully transected at 0.5-1 mm from the optic nerve head. During this procedure care is taken to avoid injury to the ophthalmic artery. Fundus examination is routinely performed immediately after axotomy and 3 days later to verify the integrity of the retinal circulation after surgery. Animals showing signs of compromised blood supply are excluded from the study.

Intravitreal injection: caspase-2 and caspase-6 inhibitors are used in this study. The inhibitors are administered by a single intravitreal injection (2 μΐ) into the vitreous chamber of the left eye of YFP mice at the time of the optic nerve injury. The intravitreal injections were made using a 10 μΐ Hamilton syringe adapted with a 32 gauge glass micro needle as described previously (Lebrun-Julien et al, 2009). Briefly, the micro needle is introduced in the superior hemisphere of the ocular globe. During this procedure care is taken to avoiding lens injury by introducing the micro needle at an angle of 45 degree through the sclera. The injection is performed over a period of 2 minutes and the needle is held still during another 2 minutes to enable the inhibitors to diffuse into the vitreous chamber. After the injection, surgical glue (Indermill, Tyco Health Care, Mansfield, MA, USA) is immediately used to seal the site of injection, avoiding any leakage.

Results: The compositions disclosed herein are tested in this animal model, and show efficacy in protection of retinal ganglion cell dendrites after axonal injury.

Example 8: Oxygen-Induced Retinopathy (OIR) Rat Model for Evaluation of Protection of Retinal Ganglion Cells following Ischemia-Reperfusion Injury

Oxygen-Induced Retinopathy (OIR) model is a relevant model for angle closure glaucoma.

Methods

Animal Model: Male Brown Norway rats (Japan SLC, Inc., Shizuoka) weighing approximately 200 to 250 g each are used in accordance with the ARVO Statement for the Use of Animals in Vision and Ophthalmic Research. Only one eye of each rat is used. The rats are anesthetized for all procedures with a mixture (1 : 1) of xylazine hydrochloride (4 mg/kg) (Bayer, Tokyo, Japan) and ketamine hydrochloride (10 mg/kg) (Sankyo, Tokyo, Japan), and the ocular surface is then anesthetized with topical instillation of 0.4% oxybuprocaine hydrochloride (Santen, Osaka, Japan). The pupils are dilated with 0.5% tropicamide and 2.5% phenylephrine hydrochloride (Santen). Ischemia-Reperfusion: The rats are placed under deep anesthesia with intramuscular injection of ketamine and xylazine. Ischemia is applied to the eye by increasing the intraocular pressure to cut off the blood supply from the retinal artery. Increased pressure is achieved by introduction of sterile saline through a 30-gauge needle that is inserted into the anterior chamber of the eye through the cornea. Each anterior chamber is cannulated with a 30-gauge infusion needle connected to a normal saline (0.9% sodium chloride) container through tubing (TI-U450P07, Terumo, Tokyo). The IOP in the cannulated eyes is raised to 90 mmHg for a period of 90 min by elevating the saline container. Intraocular pressures is measured using a rebound microtonometer designed for use on rodent eyes (TonoLab, Icare, Helsinki, Finland). Total eye ischemia is evident from the whitening of the anterior segment of the eye and the blanching of the retinal arteries on fundus examination. At the end of the ischemic period, the needle is removed from the anterior chamber, and reperfusion of the retinal vasculature is confirmed.

FG labeling: Rats are anaesthetized and sterile eye lubricant ointment is applied to prevent drying of the corneas during surgery. Head fur is shaved (from eye to ear level) and the head is fixed on the head stage by a head clamp. Operation area is disinfected with 10% povidone iodine solution followed by 70% alcohol. The point of Fluor-Gold injection is designated at a depth of 3.5 mm from the brain surface, 6.5 mm behind the bregma, 2.0 mm lateral to the midline. A hole is drilled in the skull and at the superior colliculi are injected with 2.5 micro litter of 4 % FG (Fluorochrome, Inc. 529400, Englewood, CO).

RGC counts: Labeled RGCs are counted in photographs taken from 12 areas (0.2 x 0.2 mm) of each retina situated, three in every retinal quadrant from the optic disc. The number of labeled cells in the photographs is divided by the area of the region to obtain mean densities of labeled cells per square millimeter, and the densities obtained in the 12 areas are pooled to calculate a mean RGC density per retina. Distinguishable glial cells (bright and small cells) are not counted. Cell counts are performed in a masked fashion.

Optical Coherence Tomography (OCT) retinal thickness analysis: OCT is based on low coherence interferometry and provides high-resolution cross-sectional images of the retina. Retinal thickness was measured with OCT (Cirrus OCT; Carl Zeiss Meditec, Inc., Dublin, CA). After maximal pupillary dilatation, anesthetized rats are mounted in a head holder. The optic disc is placed in the center of the OCT image, and the scanning line is aligned to pass through both the inner and outer canthi. The scan length was 5.0 25 mm in all cases. Retinal thickness is measured by OCT at 1-disc diameters from the optic disc margin in the peripheral retina with an accessory program of the OCT instrument. The mean retinal thickness of one eye is defined as the average of the three measurements. All image analyses are performed in a masked fashion.

Results: The compositions disclosed herein are tested in this animal model, and show efficacy in preservation of retinal thickness.

Example 9: Rat Axotomy Model for Evaluation of the Neuroprotective Effect of a combination of a Caspase-2 inhibitor and a Caspase-6 inhibitor

Evaluation of the neuroprotective effect of a combination of a caspase-2 inhibitor and a caspase-6 inhibitor after administration by intravitreal (IVT) is performed in Rat Axotomy

Model at two (2) weeks post injury.

Objective: to evaluate potential additive or synergistic effects of a caspase-2 inhibitor and a caspase-6 inhibitor in RGC neuroprotection in optic nerve axotomy model.

Methodology: Optic nerve axotomy is performed in adult rats. Immediately after surgery, rats receive intravitreal injections (injection volume is 5 uL) with a negative control or with the a combination of a caspase-2 inhibitor and a caspase-6 inhibitor. Second similar injections are performed into corresponding eyes at 1 week after axotomy. Evaluation of the neuroprotective effects of each of the treatments is performed by counting of FG relabeled RGC in retinal whole mounts at 2 weeks after axotomy.

Results: The compositions disclosed herein are tested in this animal model, and show efficacy in providing neuroprotection.

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Claims

CLAIMS:

1. An inhibitor of caspase-2 for use in the treatment of a disease, a disorder or an injury of the nervous system by combined, sequential or separate administration with an inhibitor of caspase-6.

2. An inhibitor of caspase-2 for use according to claim 1, wherein the nervous system is selected from the group consisting of the central nervous system (CNS) and the peripheral nervous system (PNS).

3. An inhibitor of caspase-2 for use according to claim 2, wherein the nervous system comprises the central nervous system (CNS).

4. An inhibitor of caspase-2 for use according to claim 3, wherein the disease, disorder or injury is selected from the group consisting of an ocular disease; an ocular disorder or an ocular injury; a spinal cord disease, a spinal cord disorder or a spinal cord injury; a brain disease, a brain disorder or a brain injury; and a disease, disorder or injury of the audio- vestibular system.

5. An inhibitor of caspase-2 for use according to claim 3 or claim 4, wherein the disease, disorder or injury is selected from the group consisting of a neurodegenerative disease and a neurological disorder.

6. An inhibitor of caspase-2 for use according to any one of claims 3 to 5, wherein the disease, disorder or injury is selected from the group consisting of inflammation, neurotoxicity, oxidative stress, and traumatic damage due to a tumor growth, or a combination thereof.

7. An inhibitor of caspase-2 for use according to claim 3, wherein the disease, disorder or injury of the central nervous system (CNS) is an ocular disease, an ocular disorder or an ocular injury.

8. An inhibitor of caspase-2 for use according to any one of claims 1 to 3, wherein the treatment provides neuroprotection.

9. An inhibitor of caspase-2 for use according to any one of claims 1 to 3 or 8, wherein the treatment promotes axon regeneration of neuronal cells.

10. An inhibitor of caspase-2 for use according to claim 4, wherein the treatment promotes axon regeneration of retinal neuronal cells or dorsal root ganglion cells.

11. An inhibitor of caspase-2 for use according to claim 8 or claim 9, wherein the neuroprotection is selected from the group consisting of protection of neural cells from apoptosis, promoting survival of neural cells, increasing the number of neural cell neuritis, increasing neurite cell outgrowth, promoting retinal gliosis, promoting regeneration of neural cells and increasing or stimulation of neurotrophic factors in the nervous system.

12. An inhibitor of caspase-2 for use according to any one of claims 8 to 11, wherein the neuroprotection comprises protecting neurons from death.

13. An inhibitor of caspase-2 for use in providing neuroprotection and axon regeneration of neuronal cells by combined, sequential or separate administration with an inhibitor of caspase-6.

14. An inhibitor of caspase-2 for use according to any one of claims 1 to 13, wherein the inhibitor of caspase-2 and the inhibitor of caspase-6 are independently selected from the group consisting of a small organic molecule, a protein, an antibody or fragment thereof, a peptide, a polypeptide, a peptidomimetic and a nucleic acid molecule; or a salt or prodrug thereof.

15. An inhibitor of caspase-2 for use according to claim 14, wherein at least one of the inhibitor of caspase-2 and the inhibitor of caspase-6 is a nucleic acid molecule independently selected from the group consisting of a single stranded antisense nucleic acid (ssNA), a double-stranded NA (dsNA), a small interfering NA (siNA), a short hairpin NA (shNA), a micro RNA (miRNA), an aptamer, and a ribozyme, or a salt or prodrug thereof.

16. An inhibitor of caspase-2 for use according to claim 15, wherein each of the ssNA or the dsNA independently comprises one or more of a modified nucleotide, an unmodified nucleotide, a nucleotide analogue and an unconventional moiety.

17. An inhibitor of caspase-2 for use according to claim 16, wherein at least one of the inhibitor of caspase-2 and the inhibitor of caspase-6 is a double-stranded NA (dsNA) independently selected from an unmodified double-stranded NA (dsNA) or a chemically modified double-stranded NA (dsNA); or a salt or prodrug thereof.

18. An inhibitor of caspase-2 for use according to claim 17, wherein the inhibitor of caspase-2 comprises an antisense strand sequence 5' AGGAGUUCCACAUUCUGGC 3' (SEQ ID No. 1) and a sense strand sequence 5' GCCAGAAUGUGGAACUCCU 3' (SEQ ID No. 2).

19. An inhibitor of caspase-2 for use according to any of claims 1 to 18, for combined, sequential or separate administration with a neurotrophic factor.

20. A composition comprising an inhibitor of caspase-2 and an inhibitor of caspase-6.