WO2011021221A2 - Compositions for spinal cord injury - Google Patents

Abstract

This invention relates to the use of short nucleic acid molecules that modulate RhoA kinase (RhoA) expression. The invention includes compounds, compositions and methods useful for silencing the expression and activity of the RhoA gene involved in the RhoA kinase pathway. In one embodiment, the present invention provides short nucleic acid molecules, such as siRNA, which can be used in treating, preventing, or inhibiting symptoms associated with acute spinal cord injuries.

Description

TITLE :

COMPOSITIONS FOR SPINAL CORD INJURY

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

The present application claims benefit of the filing date of Indian Provisional Patent Application No. 1885/MUM/2009 filed August 17, 2009, which is entirely incorporated herein by reference.

FIELD OF THE INVENTION:

The present invention relates to the short nucleic acid molecules, such as short interfering nucleic acid (siNA) molecules, compositions for modulating gene and protein expression, including compounds, and uses of small nucleic acid molecules to modulate Rho kinase A (Rho A) expression in spinal cord injury. The compositions and methods of the present invention have applications in spinal cord injury, either alone or in combination with other therapies.

BACKGROUND OF THE INVENTION

Spinal cord injuries (SCI) cause significant impact on quality of life and is also life threatening depending on the severity of the condition. At present there are no cure/restoration therapies available for SCI. As per the 2006 estimates, there are 2.5 million people living with various kinds disabling SCI and every year 130,000 new cases are being added. Currently the therapies available for treating SCI are aimed at controlling some body functions, such as bladder/bowel function restoration, eradicating autonomic dysreflexia, improving ^" cardiovascular functioning, and reducing spasticity, improving trunk stability, regaining normal sensation and eliminating chronic pain. The present day clinical trials in progress are aimed at only small improvements in quality of life.

Symptoms/deformities observed in SCI are due to contusion, compression, penetration or maceration leading to death of glial and neuronal cells. The events following SCI can be categorized into two different groups. These are primary spinal shock, which is acute in nature, followed by secondary events as a result of spinal shock. The majority of i functional deformities arise over a period of time (days) after SCI, and are due to secondary events. During spinal shock central grey matter fills with heavy bleeding, which eventually cuts off blood flow; oxygen supply diminishes to neuronal cells; posterior portions of spinal shock become temporarily paralyzed due to loss of communication with brain. In more severe conditions, a complete loss of reflexes and sensation in limbs will be lost. Spinal shock is a start for more devastating secondary events to take place if not treated in hours. Events such as death of glial and neuronal cells over a period of days due to induction of apoptosis result in loss of vital communications as severed axons and its neighbors get stripped of myelin sheath resulting in interruption of communication signals. This causes triggering of inflammatory immune system response. Area of destruction expands resulting in increased disability. Changes in blood flow causes damage in and around spinal cord and spreads to injured areas. The decrease in blood flow to the injured site can be tolerated during the first 1-1.5 hours by body and heart. If left untreated, self regulating capacity will turn off. Excessive flooding of neurotransmitters results in excitotoxicity which causes death of oligodendrocytes. Glial cell barrier or scar tissue prevents the regrowth of axons.

To control secondary events and limit damage caused by SCI, administration of methylprednisolone or ganglioside GMl is administered as standard of therapy. These are anti-inflammatory drugs and believed to minimize secondary damages if given within 8 hours of SCI. However, continuous death of neuronal and glial cells due to apoptosis induction and failure in re-growth of severed axons remain a major challenge to minimize the functional damages being observed. Further, myelin derived proteins are found to inhibit the regeneration of axons while scar tissue formed by glial cells and cysts due to fluid filled sacs cause obstruction of axons to penetrate or bridge. In addition, myelin associated inhibitors such as NogoA and MAG inhibit axonal re-growth. Also, small GTPase RhoA is also found to show 10 fold increase in its activity at the site of injury resulting in promoting collapse of growth cone in axonal regeneration, which acts at down stream of NogoA during signal transfer process. RhoA is a small GTPase protein known to regulate the actin cytoskeleton in the formation of stress fibers. It acts upon two known effector proteins: ROCKl (Rho- associated, coiled-coil containing protein kinase 1) and DIAPHl (diaphanous homolog 1 (Drosophila)). RhoA is part of a larger family of related proteins known as the Ras superfamily, which are proteins involved in the regulation and timing of cell division.

Rho family proteins include RhoA, RhoB, RhoC, Racl, Rac2 and Cdc42, which share more than 50% sequence identity with each other. The Rho family proteins are believed to be involved in inducing the formation of stress fibers and focal contacts in response to extracellular signals such as lysophosphatidic acid (LPA) and growth factors (A. J. Ridley & A. Hall, Cell, 70, 389-399 (1992); A. J. Ridley & A. Hall, EMBO J., 1353, 2600-2610 (1994)).

The subfamily Rho is also considered to be implicated in physiological functions associated with cytoskeletal rearrangements, such as cell morphological change (H. F. Parterson et al., J. Cell Biol., I l l, 1001-1007 (1990)), cell adhesion (Morii, N. et al., J. Biol. Chem., 267, 20921-20926 (1992); T. Tominaga et al., J. Cell Biol., 120, 1529-1537 (1993); Nusrat, A. et al., Proc. Natl. Acad. Sci. USA, 92, 10629-10633 (1995)*; Landanna, C. et al., Science, 271, 981-983 (1996), cell motility (K. Takaishi et al., Oncogene, 9, 273-279 (1994), and cytokinesis (K. Kishi et al., J. Cell Biol., 120, 1187- 1195 (1993); I. Mabuchi et al., Zygote, 1, 325-331 (1993)).

In addition, it has been suggested that the Rho is involved in the regulation of smooth muscle contraction (K. Hirata et al., J. Biol. Chem., 267, 8719-8722 (1992); M. Noda et al., FEBS Lett., 367, 246-250 (1995); M. Gong et al., Proc. Natl. Acad. Sci. USA, 93, 1340-1345 (1996)*), and the expression of phosphatidylinositol 3-kinase (PI3 kinase) (J. Zhang et al., J. Biol. Chem., 268, 22251-22254 (1993)), phosphatidylinositol 4-phosphate 5-kinase (PI 4,5-kinase) (L. D. Chong et al., Cell, 79, 507-513 (1994)) and c-fos (C. S. Hill et al., Cell, 81, 1159-1170 (1995)).

US 2005/0209147 describes peptide inhibitors of RhoA signalling that find use in the inhibition of RhoA mediated pathways involved in cancer proliferation and/or metastasis; to prevent fibrosis and wound retraction after surgical intervention (peptides block ROCK activation by RhoA); to prevent, or reduced, formation of lymphocyte syncytia during HIV infection

Chemical inhibitors of compound fasudil (CAS 103745-39-7, HA- 1077, AT-877) has been shown to be an inhibitor of Rho kinase activity (Asano et al. (1987) J. Pharmacol. Exp. Ther. 24:1033-1040). Fasudil, (or Hexahydro-l-(5-isoquinolylsulfonyl)-lH-l,4-di- azepine) has been described as the therapeutic drug of choice in treating cerebral vasospasm subsequent to subarachnoid hemorrhage (U.S. Pat. No. 6,153,608), and has been suggested for use in treatment of ischemic coronary syndrome caused by coronary artery spasm (Matsumoto et al. (2002) Circulation 105:1545-1547).

Various other inhibitors of Rho kinase have been developed, which are described in U.S. patent documents, such as: US 6218410, US 6451825, US 6586425, US 6649625, US 6673812, US 6720341, US 6720341, US 6794398, US 6844354, US 6855688, US 7199147, US 2007/0149473.

Cethrin® is a recombinant Rho antagonists comprising C3 enzymes with basic stretches of amino acids (e.g., a basic amino acid rich region) or a proline rich region added to the C3 coding sequence to facilitate the uptake thereof into tissue or cells. Cethrin's active ingredient, BA-210, is a recombinant protein that acts as a Rho GTPase antagonist to promote neuroprotection and neuroregeneration in the central nervous system (CNS). It was engineered by BioAxone to effectively penetrate into CNS tissue, where it has been clearly shown to elicit the rescue and repair of damaged neurons in preclinical animal models. To obtain Cethrin®, BA-210 is mixed with a commercially available fibrin sealant, Tisseel®, and is delivered in a single dose directly onto the dura mater of the spinal cord during decompression/stabilization surgery. Cethrin® was granted orphan drug status by the U.S. Food and Drug Administration (FDA) in December 2005.

Spinal cord injuries can result in damage to the CNS often leading to severe disabling conditions such as quadripelagia, parapelagia or even mortality. Majority of the functional disorders associated with spinal cord injuries are due to loss of neuronal cells during secondary symptoms of due to failure in regeneration of severed axons. Recent studies suggest that severed axons can regenerate, provided the inhibitors of axon regeneration can be suppressed. RhoA is one of the small GTPases that shows ten to fifteen times enhanced activity soon after injury, which is responsible for induction apoptosis and subsequent damages associated with the spinal cord injuries.

A prior study has demonstrated that Rho kinase is an important target of Rho signaling (Matsui et al (1996) EMBO J. 15: 2208-2216).

All the above cited prior art has focused on the inhibition of Rho kinase with particular selectivity to ROCK 1 and 2. In order to address a more specific Rho kinase inhibition with respect to RhoA, the present invention has designed chemically synthesized short nucleic acids that can specifically and effectively direct homology specific post transcript gene silencing and therefore can be used as highly effective, selective and potent therapeutics, with minimal side effects.

Further, the invention aims to provide the efficient delivery system for these short nucleic acids. Because siRNA are a promising tools for gene specific knockdown, the present invention has focused on inhibiting the expression levels of RhoA and tested its efficacy of its compositions in controlling some of the symptoms associated with SCI injured (SCI) rats as an animal model.

OBJECT OF THE INVENTION

It is the primary object of the present invention to provide short nucleic acid molecules for modulation of RhoA gene expression, which has better specificity.

It is the object of the present invention to provide compounds having 9-30 mer short nucleic acid molecules, such as short nucleic acid molecules having 25-27 nucleotides per strand.

It is the object of the present invention to provide short nucleic acid molecules that is site directed to a target. It is the object of the present invention to provide short nucleic acid molecules, which can provide treatment for acute spinal cord injuries.

It is the object of the present invention to provide short nucleic acid molecules, which can be used alone or in combination with other therapies for effective management of acute spinal cord injury where the apoptotic death of neurons is can be beneficial to minimize damage due to injury.

It is the object of the present invention to provide short interfering nucleic acid molecules, which can be combined with conjugates such as lipids, polymers and monoclonal antibodies.

It is the object of the present invention to provide compositions of short interfering nucleic acid molecules for efficient delivery of the said molecules.

SUMMARY OF THE INVENTION

The present disclosure provides short nucleic acid molecules, its compositions and its uses for modulation of RhoA gene expression. In related embodiments, the present invention provides Rho A-targeting short nucleic acid molecules for the treatment of acute spinal cord injuries. Such molecules may be used alone or in combination with other treatments such as co-administration of anti inflammatory siRNA or chemical drugs (such as methyl prednisilone, corticosteroids, etc.) for the management and treatment of spinal cord injuries, asthma and cancer, etc.

The present invention includes short nucleic acid molecules that are specifically targeted. In some embodiments, the short nucleic acid molecules are RNA, including siRNA towards RhoA.

In some embodiments, the present invention provides siNAs having between 19 to 30 nucleotides, between 25 and 29 nucleotides, or having 27 nucleotides, where the sequence is designed for better stability and efficacy in knockdown (i.e., reduction) of RhoA gene expression. Such siNAs can be used alone or in combination with other therapies. In certain embodiments, the siNAs may be single-stranded or double-stranded, wherein each strand has between 19 to 30 nucleotides.

The present invention provides stable compositions of siNA with or without conjugation with cholesterol. In related embodiments, the invention encompasses compounds, compositions and uses of 27-mer short interfering nucleic acid molecules in modulation of RhoA gene expression. The compounds of the present invention are useful in therapy of acute spinal cord injuries for the treatment of acute spinal cord injuries either alone or in combination with other treatments or therapies such as anti inflammatory siRNA like IL-6 or chemical drugs such as methyl prednisilone.

In one embodiment, the short nucleic acid molecules of the present invention is also a short interfering nucleic acid (siNA), a short interfering RNA (siRNA), a double stranded RNA (dsRNA), a micro RNA (μRNA), and/or a short hairpin RNA (shRNA) molecule. The short nucleic acid molecules can be unmodified or modified chemically. In the some embodiments, the present invention relates to short interfering RNA having 27 nucleotides.

In one embodiment, the nucleic acid molecule of the present invention has between 19 to 30 nucleotides, between 25 and 29 nucleotides, or 27 nucleotides. In one embodiment, the nucleic acid molecule of the present invention comprises 19-30 nucleotides complementary to RNA having an RhoA nucleic acid sequence.

In another embodiment, the invention presents a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a RhoA gene, wherein the siNA comprises an antisense region, complementary to a nucleotide sequence of the RhoA gene or a portion thereof, and a sense region substantially similar to the nucleotide sequence of the RhoA gene or a portion thereof. In one embodiment, the antisense region and the sense region each comprise about 19 to about 30 (e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, wherein the antisense region comprises at least 19 nucleotides that are complementary to nucleotides of the sense region. In one embodiment, the sense and antisense regions each comprise 25-27 nucleotides.

Nucleotides of the present invention can be chemically synthesized, expressed from a vector, or enzymatically synthesized.

In one embodiment, a siRNA molecule of the invention comprises a double stranded RNA wherein one strand of the RNA is complimentary to the RNA of Rho A. In another embodiment, a siRNA molecule of the invention comprises a double stranded RNA wherein one strand of the RNA comprises a portion of a sequence of RNA having RhoA sequence.

In one embodiment, the invention targets RhoA as set forth in GenBank Accession Number NM OO 1664. However, the present invention is not limited to nucleotides targeting one variant of RhoA, but also includes nucleotides that target RhoA-related molecules including single nucleotide polymorphisms of RhoA, RhoA homologs, and RhoA splice and transcript variants. The present invention also contemplates nucleotides that target genes involved in RhoA regulatory pathway as a means of regulating RhoA.

In other embodiments, the present invention provides compositions and methods used to regulate RhoA. RhoA may be regulated by a small nucleic acid molecule which targets RhoA directly, or by targeting molecules which regulate the RhoA pathway. Small nucleic acid molecule that target RhoA may be used alone, or in combination with other small nucleic acid molecules or small chemical molecules. In related embodiments, the targeting of RhoA is used to regulate apoptosis inhibition in neuronal cells (acute SCI), apoptosis induction in epithelial cells (cancer) and to prevent glaucoma (due to intraocular pressure) disease states that respond to modulation of RhoA expression levels in the cell.

In some embodiments, chemically synthesized siNA of 27 nucleotides in length are used to reduce expression levels of RhoA either alone or in combination with other small nucleic acid molecules directed against genes that are involved in same treatment, such as Nogo.

In one embodiment, the invention features a mammalian cell, for example a human cell or a rat neuronal cell (PC 12), comprising a small nucleic acid molecule of the invention.

The present invention features a method of down-regulating (also called "knocking down") RhoA kinase activity in a cell, comprising contacting the cell with an enzymatic nucleic acid molecule or antisense nucleic acid molecule, or other nucleic acid molecule of the invention, under conditions suitable for down-regulating of RhoA activity.

In one embodiment, the present invention also features a method of treatment of a subject having a condition associated with the level of RhoA, comprising contacting cells of the subject with the enzymatic nucleic acid molecule or antisense nucleic acid molecule or other nucleic acid molecule of the invention, under conditions suitable for the treatment. In one embodiment, a method of treatment of the invention comprises the use of one or more drug therapies under conditions suitable for said treatment.

In further related embodiments, the present invention also features a method for treatment comprising administrating a therapeutic agent, such as a brain function improving drug, use of inhibitors of Rho kinases, especially of human Rho kinases, (especially of the compounds described in the Examples), in the in vivo stimulation of nerve growth, especially of mammals, in the in vivo inhibition of scar tissue formation, especially of mammals, following damage to the brain, spinal cord or other nerves and/or in the in vivo reduction of secondary damage, especially of mammals or humans, following damage to the brain, spinal cord or other nerves, especially of humans or mammals.

In related embodiments, the present invention provides a delivery of therapeutically effective short interfering nucleic acid that modulates Rho kinase in indications such as traumatically damaged nervous system, damage from unknown causes such as multiple sclerosis, HIV dementia, Parkinson's disease, Alzheimer's disease, prion diseases or other diseases of the CNS with damaged axons and spinocerebellar ataxia 1, 2, 3, 6, 7, and 17, dentarubral-pallidoluysian atrophy, spinobulbar muscular atrophy, myotonic dystrophy and motor neuron disorders.

In another embodiment, the present invention provides compositions for efficient delivery of the short interfering nucleic acid molecules. In one preferred embodiment the present invention provides compositions comprising cholesterol conjugated 27mer siRNA.

The present invention features compositions comprising the enzymatic nucleic acid and/or antisense nucleic acid molecules of the invention in a pharmaceutically acceptable carrier.

The invention also features a method of administering to a cell, such as mammalian cell (e.g., a human cell) a nucleic acid of the invention. Such a cell can be in culture or in a mammal, such as a human. The method of administering comprises contacting the cell with the enzymatic nucleic acid molecule or antisense molecule or other nucleic acid molecule of the invention under conditions suitable for such administration. The method of administration can be in the presence of an in vivo delivery reagent, for example cholesterol conjugation of a 27mer siRNA targeted to RhoA.

Accordingly, one aspect of the present invention is an siRNA that targets a sequence selected from the group consisting of SEQ ID NO: 11; SEQ ID NO: 10; and SEQ ID NO: 9 in human RhoA mRNA of Genbank Accession number NM OO 1664.2, wherein at least one nucleotide strand of the siRNA is between 20 and 30 nucleotides in length. In one embodiment, the siRNA has a paired nucleotide sequence structure selected from the group consisting of:

(i) SEQ ID NO: 5 and SEQ ID NO: 6; (ii) SEQ ID NO: 3 and SEQ ID NO: 4; and (iii) SEQ ID NO: 1 and SEQ ID NO: 2. In another embodiment, a cholesterol moiety is conjugated to at least one nucleotide strand. In one embodiment a cholesterol moiety is conjugated to at least one of the paired nucleotide strands.

Another aspect of the present invention is a method of reducing RhoA expression in a target cell by administering any one of the siRNA molecules described herein, or designed according to the principles described herein. In one embodiment, the method of reducing RhoA expression in a target cell comprises administering an siRNA that has a paired nucleotide sequence structure selected from the group consisting of:

(i) SEQ ID NO: 5 and SEQ ID NO: 6; (ii) SEQ ID NO: 3 and SEQ ID NO: 4; and (iii) SEQ ID NO: 1 and SEQ ID NO: 2.

Another aspect of the present invention is a composition comprising a short nucleic acid molecule that modulates RhoA expression, wherein the short nucleic acid molecule is up to 30 oligonucleotides in length, and wherein the short nucleic acid molecule comprises a nucleotide sequence that is 100% complementary to at least 25 consecutive nucleotides in a sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 10, and SEQ ID NO: 9. In one embodiment, the short nucleic acid molecule comprises a sequence selected from the group consisting of:

(i) siRNA 52 comprising sense strand SEQ ID NO: 5 and antisense strand SEQ ID NO: 6;

(ii) siRNA 51 comprising sense strand SEQ ID NO: 3 and antisense strand SEQ ID NO: 4; and

(iii) siRNA 50 comprising sense strand SEQ ID NO: 1 and antisense strand SEQ ID NO: 2.

In another embodiment, a cholesterol is conjugated to at least one of the sense or antisense strands of the short nucleic acid molecule. Another aspect of the present invention is a composition comprising a short nucleic acid molecule up to 30 oligonucleotides in length that modulates RhoA expression, wherein the short nucleic acid molecule comprises a nucleotide sequence that is 100% complementary to an entire sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 10 and SEQ ID NO: 9. In one embodiment, a cholesterol is conjugated to at least one of the sense or antisense strands of the short nucleic acid molecule.

Another aspect of the present invention is a method for modulating RhoA expression in a cell, comprising contacting the cell with a short nucleic acid molecule that modulates RhoA expression, wherein the short nucleic acid molecule is up to 30 oligonucleotides in length, and wherein the short nucleic acid molecule comprises a nucleotide sequence that is 100% complementary to at least 25 consecutive nucleotides in a sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 10, and SEQ ID NO: 9. In one embodiment, the short nucleic acid molecule is selected from the group consisting of a short interfering nucleic acid (siNA), short interfering RNA (siRNA), double stranded RNA (dsRNA), micro RNA (μRNA), short hairpin RNA (shRNA), and interfering DNA (DNAi) molecules. In another embodiment, the short nucleic acid molecule comprises 21-27 nucleotides that are 100% complementary to a sequence within RhoA nucleotide sequence Genbank Accession number NM OO 1664.2. In a further embodiment, a cholesterol moiety is conjugated to at least one of the sense or antisense strands of the short nucleic acid molecule.

Another aspect of the present invention is a method for treating a spinal cord injury in an individual, comprising administering a composition, such as described above, to cells at a site of the spinal cord injury, wherein the expression of RhoA in at least one cell at the site of administration is downregulated, thereby treating the spinal cord injury in the individual. In one embodiment, at least one sequence of the short nucleic acid molecule " is conjugated to a cholesterol. In one embodiment, any of the methods described herein further comprise administering the short nucleic acid molecule to sites anterior and posterior to the site of the spinal cord injury. BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, the inventions of which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

Figure 1) RhoA knockdown in vitro by siRNA RINA52: Effect of RINA52 dosage on RhoA knockdown was estimated by transfecting cell lines HeLa (human cervical cancer cell line), HTB93 (human synovial sarcoma cell line) and PC 12 cells (rat neuronal cell line) from concentrations ranging from 0.01 nM to 100 nM with an enhancement of one log for each. Arrowheads in the figure represent 52 kDa (Tubulin) detected as internal control, and 24kDa (RhoA). As the concentration of RINA 52 was increased, there was a corresponding decrease in the expression level of RhoA. RINA52 mediated drop in RhoA expression level reached saturation at 1OnM concentration. This indicated the potency of RJNA 52 in inhibiting RhoA expression.

Figure IA: Lanel - Molecular weight marker; Lane 2 to 6 - HeLa cells treated with different concentrations of RINA52. Lane 2 - 0.01 nM , Lane 3 - 0.1 nM, Lane 4 - 1 nM ; - 1OnM; Lane 6 - 10OnM; Lane 7 - HeLa cells treated with 10 nM negative control 27mer siRNA (RINA FP targeted to EGFP); Lane 8 - HeLa untreated control cells.

Figure IB: Lane 1 - Molecular weight marker; Lane 2 - HTB93 cells treated with different concentrations of RINA 52. Lane 2 - 0.0 InM; Lane 3 - 0.InM; Lane 4 - InM; Lane 5 - 1OnM); Lane 6 - 10OnM; Lane 7 - HTB93 treated with 10 nM negative control 27mer siRNA; Lane 8 - HTB93 untreated control cells; Lane 9 - Molecular weight marker.

Figure 1C: Lane 1: PC12 untreated control cells; Lane 2: PC12 - PC12 cells treated with 10 nM negative conrol 27mer siRNA; Lane 3 and 4 - PC 12 cells treated with different concentrations of RINA52; Lane 3 - 10OnM, Lane 4 - 1OnM; Lane 5 - Molecular weight marker. Figure 2) RhoA knockdown in vitro by siRNA RINA52C: RhoA target knock down efficacy of siRNA RINA52C (cholesterol conjugated RINA52) was determined by treating cell lines A549 (adenocarcinoma human alveolar basal epithelial cells), PC 12 (rat neuronal cell line) and Neuro2A (mouse neuroblastoma cell line) with siRNA RINA52C. Arrowheads in the figure represent 52kDa Tubulin detected as internal control, and 24 kDa RhoA. Note the knockdown of RhoA expression in all the cell lines, indicating the potency of RTNA 52C. HP refers to HiPerfect transfection reagent. Respective densitometric values are indicated at the bottom of each lane.

Figure 2A: Lane 1 - Molecular weight marker; Lane 2 - A549 Untreated control; Lane 3 -A549 cells treated with 100 nM RINA52C Lane 4 - A549 cells treated with 100 nM RINA52C + HP; Lane 5 - A549 cells treated with 100 nM RINA 52 in HP,; Lane 6 - A549 cells treated with 10 nM RJNA52 in HP; Lane 7 - Molecular weight Marker

Figure 2B: Lane 1 - Molecular weight marker; Lane 2 - PC 12 cells treated with 10 nM RJNA52C; Lane 3 - PC12 cells treated with 100 nM RINA52C; Lane 4 - PC12 cells treated with 10 nM RTN A52 in HP; Lane 5 - PC 12 cells treated with 100 nM RINA52 in HP; Lane 6 - PC 12 cells treated with 10 nM negative control siRNA in HP; Lane 7 - PC 12 untreated control. cells

Figure 2C: Lane 1- Molecular weight marker; Lane 2 - Neuro2A cells treated with 10 nM RINA52C; Lane 3 - Neuro2A cells treated with 100 nM RINA52C; Lane 4 - Neuro2A cells treated with 10 nM RINA52 in HP; Lane 5 - Neuro2A cells treated with 100 nM RINA52 in HP; Lane 6 - Neuro2A cells treated with 10 nM control siRNA in HP; Lane 7 - Neuro2A untreated control cells.

Figure 3. In vivo efficacy of RINA52 on clinical parameters in a rat model for spinal cord injury (SCI).

Figure 3A) Wistar rats were inflicted spinal cord injury (SCI) by contusion method by employing standard Weight drop method. Immediately after SCI infliction, 200 micrograms of RINA 52 (n = 6) or negative control 27mer siRNA (RINA FP; Placebo, n = 4) was administered in complexation with HiPerfect transfection reagent, at the site of injury, and at the anterior and posterior regions of injury infliction at the T9 vertebra. The animals were observed for daily symptoms every day for five subsequent days, for clinical scoring on bladder control. As shown, RJNA 52 treated animals regained bladder control over placebo treated animals by the end of the fifth day of observation and reached grade 3.

Figure 3B) Wistar rats were inflicted SCI by contusion method employing standard weight drop method. Soon after SCI infliction, 200 micrograms of RJNA 52 or RINA FP (negative control RJNA) was administered as a complex with HiPerfect transfection agent at the site of injury, and at the anterior and posterior regions of injury infliction at the T9 vertebra. The animals were observed for daily symptoms every day for five subsequent days for clinical scoring on locomotion. As shown in the figure, RJNA 52 treated animals improved up on open field locomotion over placebo treated animals by the end of fifth day of observation and reached grade 3.

Figure 3C) Wistar Rats were inflicted SCI by contusion method employing standard weight drop method. Soon after SCI infliction, 200 micrograms of RJNA 52 or RJNA FP was administered in complexation with HiPerfect transfection agent at the site of injury, and at the anterior and posterior regions of injury infliction at the T9 vertebra. The animals were observed for daily symptoms every day for five subsequent days for clinical scoring on reflex action. As shown in the figure, RINA 52 treated animals recovered reflex action over placebo treated animals by the end of fifth day of observation and reached grade 3.

Figure 3D) Wistar Rats were inflicted spinal cord injury by contusion method employing standard weight drop method. As soon as SCI infliction 200 micrograms of RINA 52 or RJNA FP was administered in complexation with HiPerfect transfection agent at the site of injury, and at the anterior and posterior regions of injury infliction at the T9 vertebra. The animals were observed for daily symptoms every day for five subsequent days for clinical scoring on motor activity of the hind limbs. As shown in the figure, RJNA 52 treated animals improved upon usage of their hind limb, which was reflective of regaining motor activity in hind limbs over placebo treated animals by the end of fifth day of observation and reached grade 2. Figure 4. In vivo efficacy of RINA52C on clinical parameters in a rat model for spinal cord injury (SCI).

Figure 4A) Wistar rats were inflicted spinal cord injury by contusion method employing standard weight drop method. Following SCI, the rats were either left untreated treated (control; n = 6), treated with 200 micrograms of RINA 52 with HiPerfect transfection agent (positive control; n = 6), or 200 micrograms RINA 52C (n = 6), by administering at the site of injury, and at the anterior and posterior regions of injury infliction at the T9 vertebra. The animals were observed daily for clinical scoring on bladder control for six subsequent days. As shown in the figure, RINA 52C treated animals regained bladder control better than RINA 52. Untreated animals did not gain any bladder control.

Figure 4B) Wistar rats were inflicted spinal cord injury by contusion method employing standard weight drop method. Following SCI, the rats were either left untreated treated (control; n = 6), treated with 200 micrograms of RINA 52 with HiPerfect transfection agent (positive control; n = 6), or 200 micrograms RTNA 52C n = 6, by administering at the site of injury, and at the anterior and posterior regions of injury infliction at the T9 vertebra.. The animals were observed daily for clinical scoring on locomotion, for six subsequent days. As shown in the figure, RINA 52C treated animals improved better open field locomotion than RINA 52 by the end of sixth day. Untreated animals showed significantly lower locomotion capabilities than the two treated groups.

Figure 4C) Wistar rats were inflicted spinal cord injury by contusion method employing standard weight drop method. Following SCI, the rats were either left untreated treated (control; n = 6), treated with 200 micrograms of RTNA 52 with HiPerfect transfection agent (positive control; n = 6), or 200 micrograms RINA 52C (n = 6), by administering at the site of injury, and at the anterior and posterior regions of injury infliction at the T9 vertebra. The animals were observed daily for clinical scoring on reflex action, for six subsequent days. As shown in the figure, RINA 52C treated animals showed better reflex action from day 1 of treatment over RINA 52 treated animals. However, by the end of sixth day of observation both RINA 52C and RINA 52 had comparable reflex action abilities, with untreated showing marginal to total absence of reflex action. Figure 4D) Wistar rats were inflicted spinal cord injury by contusion method employing standard weight drop method. Following SCI, the rats were either left untreated treated (control; n = 6), treated with 200 micrograms of RINA 52 with HiPerfect transfection agent (positive control; n = 6), or 200 micrograms RINA 52C (n = 6), by administering at the site of injury, and at the anterior and posterior regions of injury infliction at the T9 vertebra. The animals were observed daily for clinical scoring on motor activity for six subsequent days. As shown in the figure, RINA 52C treated animals showed significant motor activity in hind limbs from day 1 of treatment over RINA 52 treated animals. As was seen with the reflex action, by the end of sixth day of observation both RINA 52C and RINA 52 had comparable motor activity abilities in hind limbs over untreated treated animals.

Figure 5. In vivo target knockdown efficacy of RINA52 on SCI rats. Spinal cord injured Wistar rats were administered either RINA 52 or RINA FP (placebo), complexed with HiPerfect transfection reagent, at the site of injury, and at the anterior and posterior regions of injury infliction at the T9 vertebra. At the end of 48 h of administration of siRNA, spinal cords were recovered from the each animal and segregated as anterior to SCI, at the site SCI and posterior to SCI. Total protein lysates were isolated from all the regions of both RINA 52 and RINA FP treated animals and subjected to Western blot analysis using antibodies specific to Tubulin (52 kDa) and RhoA (24 kDa) Lane 1 - Molecular weight marker; Lane 2 - RINA 52 treated (posterior to injury); Lane 3 - RTNA 52 treated (anterior to injury ); Lane 4 - RINA 52 treated (site of injury); Lane 5 - RINA FP treated (48 h posterior to injury); Lane 6 - RTNA FP treated (48 h anterior to injury); Lane 7 - RINA FP treated at the site of injury (48 h); Lane 8 - A431 protein lysate as a positive control for RhoA identification. Result show that RhoA was knocked down at the site of injury rather than at the anterior or posterior regions of SCI in RINA52 treated animals as compared to that of RTNA FP treated animals. These results suggest that clinical improvements observed in RTNA52 treated animals correlate with knockdown of RhoA.

Figure 6. In vivo target knockdown efficacy of RINA52C on SCI rats. Figure 6A and B). Wistar Rats were inflicted spinal cord injury by contusion method employing standard weight drop method. The spinal cord injured rats were either left untreated (n = 3) or administered RINA 52C at at the site of injury, and at the anterior and posterior regions of injury infliction at the T9 vertebra (n=3). At the end of the monitoring period of 6 days (ref: clinical scoring data in Figure 4 above), the spinal cords were recovered from the each animal and segregated as cranial to SCI, site of SCI and caudal to SCI. The samples were immediately stored in liquid nitrogen. Total protein was isolated from all the regions of RINA 52C treated animals, untreated injured animals and uninjured normal (control) animals and subjected to Western blot analysis using antibodies specific to RhoA and Tubulin. Result showed that RhoA levels were elevated upon spinal cord injury. The result also indicated that the elevated RhoA was knocked down by RTNA 52C treatment, at all the three sites tested, relative to the untreated control. The Western blot analysis results are shown for two out of three 52C treated animals (Figure 6 A— animal # 1 and Figure 6B - animal # 2). These results clearly correlate with our clinical observations of improved bladder control, BBB locomotion; reflex action and motor activity in the hind limbs in RINA52 treated animals and are due to knockdown of RhoA in the spinal cord. Apparently RINA52C molecules were taken up by spinal cord cells, resulting in effective clinical improvement arising due to target oriented delivery of siRNA. Lane 1 - Molecular weight marker; Lane 2 - Normal uninjured animals hours posterior to injury; Lane 3 - Untreated cranial portion, Lane 4 - Untreated site of injury; Lane 5 - Untreated caudal portion; Lane 6 - RINA 52C treated cranial portion; Lane 7 - RINA 52C treated site of injury; Lane 8 - RINA 52C treated caudal portion.

Figure 6C) Graphical representation of densitometry values from the two independent western blot experiments with standard error.

DETAILED DESCRIPTION OF THE INVENTION

Definitions: The term "short nucleic acid molecule" refers to any nucleic acid molecule capable of modulating gene expression. The terms "short interfering nucleic acid", "siNA" or "siNA molecules, "short interfering nucleic acid molecule", "short interfering oligonucleotide molecule" refer to any nucleic acid molecule capable of inhibiting, down- regulating or knocking down gene expression.

Typically, short interfering nucleic acid molecules are composed primarily of RNA, and may be referred to as "short interfering RNA" or "siRNA." A siNA may, however, include nucleotides other than RNA, such as in DNAi (interfering DNA), or other modified bases. Thus, the term "RNA" as used herein means a molecule comprising at least one ribonucleotide residue and includes double stranded RNA, single stranded RNA, isolated RNA, partially purified, pure or synthetic RNA, recombinantly produced RNA, as well as altered RNA such as analogs or analogs of naturally occurring RNA

The term "modulate" or "modulates" means that the expression of the gene or level of RNA molecule or equivalent RNA molecules encoding one or more protein or protein subunits or peptides, or activity of one or more protein subunits or peptides is up regulated or down regulated such that the expression, level, or activity is greater than or less than that observed in the absence of the modulator. The term "modulate" includes "inhibit."

The terms "inhibition" (or "inhibit"), "down-regulation" (or "down-regulate") or "knockdown" of a gene means that expression of a gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits, is reduced below that observed in the absence of an inhibitory nucleic acid molecule (e.g., siNA) of the present invention, hi one embodiment, inhibition or down-regulation observed in the presence of one or more siNA molecules is greater than inhibition or down-regulation observed in the absence of the siNA(s) or in the presence of, for example, a siNA molecule with a scrambled sequence or with mismatches. Likewise, in one embodiment, expression of a gene or protein is "knocked down" in the presence of one or more siNA molecules, as compared to gene or protein expression observed in absence of the siNA molecule(s), or in the presence of, for example, a siNA molecule with a scrambled sequence or with mismatches.

The term "mock treated" refers to cells treated with an non specific siRNA which does not target any known genes in the mammalian cells. Here in this study we have used "RTNA FP" as a mock siRNA, which encodes for EGFP protein._. RINA FP was custom synthesized by Qiagen.

The term "gene" as used herein means a nucleic acid that encodes a RNA sequence including but not limited to structural genes encoding a polypeptide.

The term "RhoA" as used herein refers to any RhoA protein, peptide, or polypeptide or a derivative thereof, such as encoded by Genbank Accession number NM OO 1664, having Rho Kinase A or RhoA activity. The term RhoA also refers to nucleic acid sequences encoding any RhoA protein, peptide, polypeptide, or polypeptide having isoforms, mutant genes, splice variants or polymorphisms, having RhoA activity.

The term "target nucleic acid" as used herein means any nucleic acid sequence whose expression or activity is to be modulated. The target nucleic acid can be DNA or RNA.

The term "sense region" as used herein with regard to a siNA of the present invention refers to a nucleotide sequence of a siNA molecule that is complementary to an antisense region of the siNA molecule. In addition, the sense region of a small nucleic acid molecule can comprise a nucleic acid sequence having homology with a target nucleic acid sequence.

The term "antisense region" as used herein with regard to a siNA of the present invention means a nucleotide sequence of a siNA molecule that is complementary to a target nucleic acid sequence. It can also comprise a nucleic acid sequence that is complementary to a sense region of a siNA molecule. The term "complementarity" or "complementary" as used herein means that a nucleic acid sequence can form hydrogen bond(s) with another nucleic acid sequence. A nucleic acid molecule comprising two or more nucleic acids may be partially or completely (100%) complementary to another nucleic acid molecule, for example, with regard to corresponding nucleic acids that are capable of forming a double stranded molecule. A percent "complementarity" or "complementary" indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds with a second nucleic acid sequence. For instance, a first sequence is 95% complementary to a second sequence if 19 out of 20 contiguous nucleotides in the first sequence form hydrogen bonds with 19 out of 20 contiguous nucleotides in the second sequence. "Completely complementary" means that all contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. For example, a 23-mer nucleic acid may be completely (100%) complementary to a 27-mer nucleic acid with regard to 23 contiguous nucleic acids.

The term "brain function disorder" includes psychotic condition due to cerebral hemorrhage, cerebral thrombus, cerebral embolus, subarachnoid hemorrhage, transient cerebral ischemic stroke, hypertensive encephalopathy, cerebral arteriosclerosis, subdural hematoma, extradural hematoma, cerebral hypoxia, cerebral edema, cerebritis, cerebral tumor, external injuiy in head, mental disease, metabolite poisoning, drug poisoning, temporal respiratory arrest, deep anesthesia during operation, physical disorder and the like, and sequelae, decreased attention, hyperactivity, logopathy, delayed mental development, lethe, dementia (inclusive of wandering, nocturnal delirium, aggressive behavior and the like associated with dementia) caused by the above-mentioned diseases.

The present invention relates to short nucleic acid molecules, its compositions and it uses for modulation of RhoA gene expression. In related embodiments, the present invention provides RhoA-targeting short nucleic acid molecules for the treatment of acute spinal cord injuries. Such molecules may be used alone or in combination with other treatments for the management and treatment of indications related to central nervous system such as spinal cord injuries, peripheral nervous system injuries etc. Further, according to literature, biomatrices could be especially important in the treatment of transaction or laceration types of SCI, when a lesion gap has to be bridged - acting as carriers for therapeutic agents, such as neurotrophic factors or stem cells.

Cholesterol conjugation of our 27mer siRNA, RINA 52 (viz., RINA 52C) was highly efficient in specifically blocking RhoA expression in cultured cell lines as well as in SCI rats. To our knowledge, this is the first time demonstration that a 27mer siRNA targeted to RhoA has in vivo efficacy in a SCI animal model. To our knowledge, this is also the first time demonstration of in vitro and in vivo knockdown of RhoA by a 27mer siRNA at the protein level.

The present invention proves that inhibition of expression levels of RhoA by siRNA can result in enhancement of functional recovery in spinal cord injured rats.

EXAMPLES.

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples, which follow, represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Antibodies used for immunoblotting included anti-RhoA mouse monoclonal antibody and anti-α-tubulin mouse monoclonal antibody, and were purchased from Abeam. Nitrocellulose membranes were obtained from Pall Life Sciences and electrophoresis supplies were purchased from Bio-Rad Laboratories. All other chemicals were purchased from Sigma Chemical Co. unless otherwise specified.

Design of siRNA The present invention provides short interfering nucleic acid (siNA) molecules and their uses in modulation of Rho gene expression. The main features of the studies conducted are as follows:

1. Design of siNA.

2. Preparation of siNA

3. Efficacy testing of the compounds

4. Comparative data of siRNA having 21, 22, and 27 nucleotides

5. Potency evaluation in an animal model

All siRNAs were designed in-house (see Tables 1 and 2 below) and custom synthesized from Qiagen. The design of suitable siNA involved the design of the siRNA with 21, 23, and 27 nucleotides for modulation of Rho, without chemical modification. The Rho target genes were screened for accessible sites and siRNA was synthesized considering the open reading frame (ORF) sequences of Rho. The following general requirements were considered in siNA design:

i. No runs of four or more A, T, G, or U in a row

ii. The following sequences were avoided, as they can induce an interferon response.

A) 5'-UGUGU-3' and B) 5'-GUCCUUCAA-S' From literature it is known that these sequences are known to induce Interferon response. So we avoided these sequences when designing siRNAs.

iii. The first 200 bases were omitted from the start codon to avoid binding to

regulatory element.

iv. Each siNA duplex was checked in silico to avoid silencing of off-target effects made on BLAST search considering the following parameters:

A. Low complexity filtering was removed to avoid insignificance by BLAST resulting in limited or no query sequencer.

B. The word size was set to 7 letters, the minimal value algorithm

C. Expected value threshold was set at 1000 to avoid the probability of short sequence occurrence.

Identification of target sites:

siRNA and their respective locations on Rho A gene are indicated in the Table 1 and 2. Table 1: Target ORF sequences of RhoA for siRNA synthesis

Table 2: siRNA synthesized with end modifications for RhoA gene.

RINA Duplex sequence with overhangs

Duplex

50 SENSE 5'-CCUGAAGAAGGCAGAGAUAUGGCdAdA-S' (SEQ ID NO: 1) ANTISENSE 5'-UUGCCAUAUCUCUGCCUUCUUCAGGUU-S' (SEQ ID NO: 2)

51 SENSE 5'-GACCAAAGAUGGAGUGAGAGAGGdTdT-S' (SEQ ID NO: 3) ANTISENSE 5'-AACCUCUCUCACUCCAUCUUUGGUCUU-S ' (SEQ ID NO: 4)

52 SENSE 5'-GAAUUAGGCUGU AACUACUUU AUdAdA-3' (SEQ ID NO: 5) ANTSENSE 5'-UUAUAAAGUAGUUACAGCCUAAUUCAC-S ' (SEQ ID NO: 6)

*FP SENSE 5'-GCG ACG UAA ACG GCC ACA AGT TCA G-3' (SEQ ID NO: 7)

ANTISENSE 5'-CUG AAC UUG UGG CCG UUU ACG UCG CCG-3' (SEQ ID NO: 8)

* FP RINA was used as a negative control = "negative RINA" in this and other experiments presented herein. The FP RINA was designed against EGFP. RINA 50, 51 and 52 were screened for their relative RhoA knock down efficiencies in culured cell lines by means of Western blot analysis using Tubulin as an internal control. RINA 52 gave the best RhoA knock down efficiency (data not shown), and was chosen as the siRNA for further experiments.

EXAMPLE 2: EXPRESSION ANALYSIS OF RHOA Cell culture

HeLa (Cervical cancer) HTB93 (synovial sarcoma) and PC3 (prostate cancer) cells were purchased from ATCC and maintained in RJPMI 1640 medium supplemented with 10% FBS. Subconfluent cultures not exceeding 10 passages were used in experiments. All cell lines were maintained at 37⁰C in 5% CO₂ in a humidified incubator. siRNA Transfection

Cells were seeded in 24-well plates at 20,000 cells/well and transfected with 1OnM siRNA, unless otherwise specified, using Hiperfect transfection reagent (Qiagen) following the protocol recommended by the manufacturer. All experiments were performed after 72 h of transfection unless otherwise specified.

The transfection efficiency of cells was determined by Cy3 labeled RINA FP. RINA FP was labeled with Cy3, a fluorescent dye following protocol of manufacturer silencer Cy3 labeling kit (Ambion). Cells at the end of 24 h of transfection were trypsinized and counted at 5 random fields under fluorescent microscope to determine the percent of cells transfected. HeLa, HTB-93 and PC3 cell lines were used.

Transfection of siRNA to the cells could be achieved successfully with efficiencies ranging from 85% - 97% depending on the cell line used. Further, the cholesterol conjugated siRNA alone without having any transfection agent are found to show transfection efficiency of equivalent to that of the Hiperfect transfection agent, as shown in Table 4. The exact experiment done was as follows: siRNA and Cholesterol conjugated siRNA were labeled using Cy3 labelling kit ( Ambion ). The cell lines listed in Table 4 were transfected using these siRNAs and transfection efficiency was determined by monitoring the cells under a Fluorescence microscope after 24 hours. In addition, our observations at different time points under fluorescent microscope showed that indeed the cholesterol conjugated siRNA were stable for longer periods of time over unconjugated siRNA intracellularly. Table 4. siRNA transfection efficiency ( determined 24 hours post transfection ) as determined by fluorescence microscopy with Cy3 labeled siRNA

* Cells transfected with cholesterol conjugated siRNA

Western blotting

Total cellular protein was isolated using Mammalian protein extraction reagent (Pierce) and estimated using Biorad protein assay (Bio-Rad Laboratories). Equal amounts of protein were loaded and separated by SDS-PAGE and western blot analysis was carried out. Specifically bound primary antibodies were detected with alkaline phosphatase- coupled secondary antibodies.

EXAMPLE 4: TESTING OF EFFICACY

The present invention establishes that inhibition of RhoA expression can result in enhanced recovery of functionality, such as bladder control, locomotion and reflex action, in SCI animals.

Determination of siRNA efficacy

The efficiency of siRNA designed (RINA 50, RINA 51 and RINA 52) was determined by employing protein blots and measuring the quantity of decrease in phosphorylated ROCK2, a down stream target of RhoA protein obtained in HeLa cells at 1OnM concentration. siRNA designed against EGFP was used as a negative control. And also total ROCK2 was determined to know non specific knockdown of ROCK2. Effect ofsiRNA concentration on RhoΛ knockdown

Cell lines HeLa, HTB-93 and PC 12 cells were transfected with RINA 52 at concentrations ranging from 0.001 nM to 100 nM with an increment of one log scale for each. At the end of 72 h cells were harvested and the decrease in RhoA expression levels with the increase in the siRNA concentration was determined by protein blot analysis. Quantitative real-time PCR

Total RNA was isolated using the RNeasy mini kit (Qiagen) following the manufacturer's instructions. Complementary DNA was prepared from 2μg of RNA using the High capacity cDNA archive kit (Applied Biosystems) according to the manufacturer's instructions. TaqMan real-time PCR reaction was set up on an Applied Biosystems 7500 system using β-actin as an endogenous control for normalization as described previously (26). All primers and probes are represented in Table 3.

Table 3: Primer and Probes used for quantitative Real Time PCR

RINA 52 acts as a siRNA rather as miRNA The cell lines (HeLa, PC3 and HTB-93) transfected with RINA 52, as analyzed by quantitative Real time PCR, showed decrease in mRNA quantity levels over RINA FP treated or untreated cells as shown in the Table 5. The decrease in mRNA levels with the decrease in protein quantity indicates that RINA 52 is indeed acting as an siRNA rather miRNA.

Table 5. Percent decrease in expression levels of RhoA target gene at 72 hours post transfection as determined by Real Time PCR

RINA 52 shows dose dependent decrease in expression of RhoA protein

With the increase in the concentration of siRNA from 0.01 nM to 100 nM with an increment of one log each, there was a corresponding decrease in the expression levels of RhoA protein as shown in the Figure 2. Of the cell lines tested, HeLa showed more than 95% decrease in protein expression (Figure 2A), while at 100 nM no detectable amounts of protein expressed by Western blot analysis. However, significant quantities of decrease in RhoA expression were observed in HTB-93 cells at 100 nM of RINA52 concentration (Figure 2B).

Determination of Interferon response

Stress responses may affect general cellular protein expression levels and can confuse the interpretation of data obtained from RNAi experiments. To validate whether the gene specific knockdown is due to siRNA or due to a siRNA induced non-specific interferon stress response, relative expression levels of four genes involved in the interferon response in ARPE-19 cells, i.e., OASl, OAS2, MXl, ISGF3g, were evaluated at 24 hours post transfection using Interferon response detection kit (System Biosciences cat no. SI300A-1) following the manufacturer's protocol.

Transfection of cells with RJNΛ52 did not elicite significant type I or type II interferon response

Among the three RhoA specific siRNAs used in this study (RJNA50, RINA 51 and RINA52), RINA52 elicited the least interferon response following transfection into ARPE- 19 cells. Result are shown in Table 6. Results indicate that RINA 52 may be particularly well suited for therapeutic applications.

Table 6. Lack of interferon response induced by RhoA siRNA as seen in reverse transcriptase PCR analysis

"UT" = untreated. Densitometric ratios have been deduced from densitometric values obtained using a Kodak densitometric analysis software from bands representative to each gene mentioned above with respect to an internal control β-actin, as visualized on an agarose gel.

Effect ofRINA 52 and RINA 52C on RhoA knockdown

Different cell lines (see below) were transfected with RINA 52 ( PC 12, HeLa and HTB93 ) or RINA 52C ( A549, PC12 and Neuro2A ), a 3^{^} cholesterol conjugated form of RINA 52 at concentrations 10 nM and 100 nM or 10OnM. At the end of 72 hours, cells were harvested and the decrease in RhoA expression levels was determined by Western blot analysis using RhoA specific monoclonal antibody ( 1 :200 dilution in TBST buffer ) purchased from Santacruz Biotech and Tubulin specific antibody from Sigma ( 1 :2000 dilution in TBST ) . Secondary antibody used was anti mouse gamma chain specific ALP conjugated antibody procured from Sigma ( 1 :10000 dilution in TBST ). Conditions used for western blot analysis were according to Maniatis et al. Results showed RNAi mediated specific knockdown of RhoA in these cells. See Figures 1 and 2 for details.

Animal model.

Spinal cord injury was inflicted in 24 male 6-8 weeks old Wister rats following methods approved by the Institutional Animal Ethics Committee (IAEC). (ref: Metz GA, Curt A, van de Meent H, Klusman I, Schwab ME, Dietz V, 2000. Validation of the weight-drop contusion model in rats: a comparative study of human spinal cord injury. J Neurotrauma. Jan; 17(1): 1-17). The protocol followed was as follows - Animals were anesthetized by administering a combination of ketamine hydrochloride (lOOmg/kg body weight) and Xylazine (10 mg/kg body weight). Fur was shaved off from ventral surface of the animal using electronic trimmer. Using sterile scalpel, an incision of about 1 cm was made at T9 vertebrae. The vertebra was lesioned by using a standardized weight-drop injury device.

In-vivo efficacy of RINA 52 in improving functional recovery of SCI Rats

The SCI rats were segregated into different groups and either left untreated or treated by injecting 200 micrograms of target specific siRNA or placebo control directly at the site of injury, as well as anterior and posterior to the site of injury (for details on specific experiments, see Description of Drawings given above). Following treatment, the muscles were sutured and the skin was closed with surgical clamps. At the site of incision, Betadine was applied while administering Oxytetracycline hydrochloride. Post surgical care was done for five days every day by administration of Oxytetracycline and application of Betadine at surgical site.

Clinical scorings were made every day for 6 days following a "BBB" (Basso, Beattie and Brasnahan) scoring system, while taking into account the general health of the animals. Basso DM, Beattie MS, Bresnahan JC, A sensitive and reliable locomotor rating scale for open field testing in rats, J. Neurotrauma, 12:1-21 (1995); Basso DM, Beattie MS, Bresnahan JC, Graded histological and locomotor outcomes after spinal cord contusion using the NYU weight-drop device versus transaction, Exp. Neurol., 139:244-256 (1996). The animals in each group were observed for behavioral changes up to 6 days from the date of inflicting injury following the BBB scoring. The parameters included for this analysis are 1. Bladder control; 2. Open Field locomotion in NBO hood; 3. Reflex action in hind limb; 4. Motor activity in hind limb. The animals were clinically scored for each of the above parameters during this observation period. The scoring was done on a scale of 1 - 4 where 1 = Bad: no bladder control; showing lack of motility; no reflex action and inability to use hind limbs. 2 = Good: partial recovery of bladder control; able to move over a short distance; reflex activity in 1 hind limb; motor activity in atleast one hind limb. 3 = Better: achieved reasonably good control over bladder; shows locomotion atleast by dragging over while exploring often; utilizing hind limbs either to support or coordinate locomotion functions with fore limbs. 4 = Best: normal balder function; normal locomotion; complete reflex action; used both the hind limbs normally.

A) Bladder control- Of all the animals treated with RINA52, 33% of the animals improved to grade 2, while 67% improved to grade 3, whereas none of the animals could recover bladder control in placebo treated group at the end of five days after treatment as shown in Figure 3A. Similarly, a dramatic improvement in bladder control was observed in all the SCI rats treated with RINA 52C, as shown in Figure 4A.

B) Open field locomotion - Animals were left in a transparent plexi box with a blotting paper to protect from skidding. Each animal was observed for a period of 90 seconds. Of all the animals tested, all RINA52 treated animals reached grade 3, while only 50% of placebo treated animals reached grade 3, while remaining animals stayed at grade 2 only, as shown in Figure 3B at the end of five days of observations. Similarly, a dramatic improvement in locomotion was observed in all SCI rats treated with RINA 52C, as shown in Figure 4B.

C) Reflex action in hind limbs - Animals treated with either RINA52 or placebo were subjected to pinching sensation and observed the ability to retract the hind limbs. Of the all RINA52 treated animals, 67% of the animals reached grade 2, where there was a delayed response of reflex action, while there was no reflex action noted in placebo treated group (Figure 3C). A similar improvement in reflex action was also observed in the SCI rats treated with RINA 52C, as shown in Figure 4C.

D) Motor activity in Hind limbs - The usage of hind limbs by SCI inflicted animals was observed during NBO hood locomotion test, as well as during grid walking. The RJNA52 treated (but not placebo treated) animals reach grade 2 for motor functionality at the end of five days of observations, as indicated in Figure 3D. A similar improvement in motor activity was also observed in the SCI rats treated with RTNA 52C, as shown in Figure 4D. Analysis of target knock down was done at 48 h after siRNA treatment (for the experiment to determine the in vivo efficacy of RTNA 52) and 6 days after siRNA treatment (for the experiment to determine the in vivo efficacy of RINA 52C). For this purpose, three animals from each group were sacrificed and the spinal cords were collected from the different sites of the spinal cord (SC), viz., anterior to injured site, at the site of injury and posterior to the injured site. The tissues collected were snap frozen in liquid nitrogen and were analyzed for RhoA knock down levels by Western blot analysis.

As shown in Figure 5, administration of RJNA 52 caused decrease in the RhoA expression only at the injured site rather at anterior or posterior to the spinal cord injury. These results indicated that RINA52 could be efficiently taken up by spinal cords only at the site of injury rather at the rest of the uninjured portions. Furthermore, the decrease in expression levels of RhoA could be attributed to the improving functionality of the SCI animals over placebo treated group since they did not show any decrease in expression levels of the RhoA.

As shown in Figures 6A, B and C, RTNA 52C had an even more pronounced in vivo RhoA knock down efficacy in SCI rats than RJNA 52 because all the three sites examined (site of injury, anterior side of the site of injury and posterior side of the site of injury), showed a reduction in RhoA levels. This result was validated in two independent animals. To our knowledge, this is the first time demonstration of in vivo efficacy of cholesterol conjugated 27mer siRNA against spinal cord injury, at the protein level.

Statistical analysis

Paired two-tailed student t-test, assuming equal variance, was performed and the P value was calculated for each experiment. For all experiments with error bars, standard deviation was calculated to indicate the variation.

All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are chemically or physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. Furthermore, because RhoA is known to be involved in several other indications (e.g., cancer, diabetic retinopathy, etc.), the methods and compositions described in this invention, may also have applications in the context of these other indications.

Claims

WE CLAIM:

•1. An siRNA that targets a sequence selected from the group consisting of SEQ ID NO: 1 1, SEQ ID NO: 10 and SEQ ID NO: 9 in human RhoA mRNA of Genbank Accession number NM OO 1664.2, wherein at least one nucleotide strand of the siRNA is between 20 and 30 nucleotides in length.

2. The siRNA of claim 1, wherein the siRNA has a paired nucleotide sequence structure selected from the group consisting of:

(i) SEQ ID NO: 5 and SEQ ID NO: 6; (ii) SEQ ID NO: 3 and SEQ ID NO: 4; and (iii) SEQ ID NO: 1 and SEQ ID NO: 2.

3. The siRNA of claim 1, wherein a cholesterol moiety is conjugated to at least one nucleotide strand.

4. The siRNA of claim 2, wherein a cholesterol moiety is conjugated to at least one of the paired nucleotide strands.

5. A method of reducing RhoA expression in a target cell by administering the siRNA of claim 1.

6. A method of reducing RhoA expression in a target cell by administering the siRNA of claim 2.

7. A composition comprising a short nucleic acid molecule that modulates RhoA expression, wherein the short nucleic acid molecule is up to 30 oligonucleotides in length, and wherein the short nucleic acid molecule comprises a nucleotide sequence that is 100% complementary to at least 25 consecutive nucleotides in a sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 10, and SEQ ID NO: 9.

8. The composition of claim 7, wherein the short nucleic acid molecule comprises a sequence selected from the group consisting of: (i) siRNA 52 comprising sense strand SEQ ID NO: 5 and antisense strand SEQ ID NO: 6;

(ii) siRNA 51 comprising sense strand SEQ ID NO: 3 and antisense strand SEQ ID NO: 4; and

(iii) siRNA 50 comprising sense strand SEQ ID NO: 1 and antisense strand SEQ ID NO: 2.

9. The composition of claim 8, wherein a cholesterol is conjugated to at least one of the sense or antisense strands of the short nucleic acid molecule.

10. A composition, comprising a short nucleic acid molecule up to 30 oligonucleotides in length that modulates RhoA expression, wherein the short nucleic acid molecule comprises a nucleotide sequence that is 100% complementary to an entire sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 10 and SEQ ID NO: 9.

11. The composition of claim 10, wherein a cholesterol is conjugated to at least one of the sense or antisense strands of the short nucleic acid molecule.

12. A method for modulating RhoA expression in a cell, comprising contacting the cell with a short nucleic acid molecule that modulates RhoA expression, wherein the short nucleic acid molecule is up to 30 oligonucleotides in length, and wherein the short nucleic acid molecule comprises a nucleotide sequence that is 100% complementary to at least 25 consecutive nucleotides in a sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 10, and SEQ ID NO: 9.

13. The method of claim 12, wherein the short nucleic acid molecule is selected from the group consisting of a short interfering nucleic acid (siNA), short interfering RNA

(siRNA), double stranded RNA (dsRNA), micro RNA (μRNA), short hairpin RNA (shRNA), and interfering DNA (DNAi) molecules.

14. The method of claim 12, wherein the short nucleic acid molecule comprises 21-27 nucleotides that are 100% complementary to a sequence within RhoA nucleotide sequence Genbank Accession number NM_001664.2.

15. The method of claim 12, wherein a cholesterol is conjugated to at least one of the sense or antisense strands of the short nucleic acid molecule.

16. A method for treating a spinal cord injury in an individual, comprising administering the composition of claim 8 to cells at a site of the spinal cord injury, wherein the expression of RhoA in at least one cell at the site of administration is downregulated, thereby treating the spinal cord injury in the individual.

17. The method of claim 16, wherein at least one sequence of the short nucleic acid molecule is conjugated to a cholesterol.

18. The method of claim 17, further comprising administering the short nucleic acid molecule to sites anterior and posterior to the site of the spinal cord injury.

19. The siRNA that targets a sequence in human RhoA mRNA of Genbank Accession number NM__001664.2, its compositions and methods for use in spinal cord injury as claimed above exemplified herein substantially in the examples and figures.