WO2007113784A2 - Methods for modulating the levels and effects of thyrotropin-releasing hormone (trh) and trh-related petides - Google Patents

Abstract

The invention provides novel methods for modulating the levels and effects of TRH and/or TRH-like peptides. These include agents for suppressing TRH-DE expression in cells, in tissues and/or in animals, such as RNAi and ribozymes. The invention has multiple applications inter alia in the generation of novel therapies for disorders related to disturbances in TRH, TRH-related peptides and TRH-DE; to modulate physiological processes; as a tool(s) for probing biological processes and mechanisms and/or as a means of generating transgenic animals.

Description

Title

Methods for Modulating the Levels and Effects of Thyrotropin-Releasing Hormone (TRH) and

TRH-related Peptides

Field of the Invention

The present invention relates to novel agents that increase the bioavailability of thyrotropin- releasing hormone (TRH). In particular, the invention relates to compounds that suppress expression of the gene encoding TRH-degrading ectoenzyme (TRH-DE) (EC 3.4.19.6), also known as pyroglutamyl aminopeptidase II (PAP-II, PP-II). The invention also relates to methods for modulating the levels and actions of thyrotropin-releasing hormone (TRH). As a result, compounds of the invention find potential therapeutic application in the field of medicine, particularly, but not limited to, conditions involving neuronal cell injury and disturbances in neurobiological function. They also have application as research tools for studying inter alia the signalling pathway and biological functions of TRH, TRH-DE and TRH receptors.

Peptide signalling with TRH involves a tripartite system: the TRH peptide, the THR receptor(s) and an inactivation system comprising TRH degrading ectoenzyme (TRH-DE), EC 3.4.19.6, also known as pyroglutamyl aminopeptidase II (PAP-II, PP-II)). Modulation of the levels and actions of TRH may find therapeutic application in the field of medicine particularly in the treatment of brain and spinal injuries and central nervous system (CNS) disorders and/or in the treatment of tissues where TRH plays a functional role. They may also have application as tools for studying the biological functions of the peptides, their receptor(s) and their inactivating enzymes.

TRH has the structure:

L-pyroglutamyl-L-histidyl-L-prolineamide (Glp-His-ProNH₂) (Kelly 1995).

The nomenclature of Schechter and Berger (Schechter and Berger 1967) is used to describe the positions of the peptide substrate residues (P) relative to the scissile Pi-Pi' bond and the corresponding subsets (S) in the active site of the enzyme. In other literature, the right portion of the molecule is called the "prolineamide" or "C-terminal" portion; the centre portion of the molecule is called the "histidyl" portion; and the left portion of the molecule is called the "pyroglutamyl", "COOH-terminal" or "N-terminal" portion. Background of the invention

The effects of CNS disorders are varied and can include fatal or life threatening consequences, impairment of motor function, stroke, spinocerebellar degeneration, memory loss, chronic and acute neurodegeneration, disorders of consciousness and disorders of mood. Effective methods of repair or cures for CNS disorders are extremely limited, and to date most treatments seek to ameliorate the symptoms rather than address underlying defects.

In recent years, much research has focused on identifying many of the components in the CNS associated with signaling, degeneration, neuroprotection and repair. TRH is a naturally occurring neuroactive peptide with multiple actions in the CNS that have been shown to be beneficial in the treatment of certain CNS disorders, including brain and spinal injury, stroke, spinocerebellar degeneration, memory loss, disorders of consciousness, spinal cord pain transmission and epilepsy (Sobue et al. 1980; Faden and Salzman 1992; Kelly 1995, Horita 1998; Nillni et al. 1999; Vetulani and Nalepa 2000; Kubek and Garg 2002; Gary et al. 2003) TRH is also believed to play a role in peripheral NS (PNS) function (Nillni et al. 1999) and hence TRH-based therapies may have relevance to PNS-related disorders. Recent literature highlights a growing recognition of the breadth of TRH functions and the potential widespread clinical applicability of this remarkable peptide (Gary et al. 2003). For example, it has been suggested recently that TRH may function as a core homeostatic regulator within four integrated CNS systems and as such may have extensive involvement and therapeutic application in human illnesses associated with disturbances in neurobiological function, including conditions as diverse as jetlag, attention deficit/hyperactive disorder and depression (Gary et al. 2003). In addition, there is growing evidence for the beneficial effects of central TRH in feeding disorders, particularly obesity (Steward et al. 2003). Other researchers strongly suggest a role for TRH in the physiology and treatment of mood disorders (Sharma et al. 2001). Luo et al. suggest that TRH may function as an endogenous neuroprotectant and that low levels of TRH found in the hippocampus of Alzheimer's patients may contribute to the pathogenesis of this disorder (Luo et al. 2002).

With respect to epilepsy, studies show TRH to be efficacious in treating patients with intractable epilepsy and indicate that TRH may represent a new class of antiepileptic (Kubek and Garg 2002). TRH and TRH analogues have been shown to have beneficial effects in animal models of motor dysfunction (Sobue et al. 1980; Matsui et al. 1996) and the only TRH-based therapeutic to reach the market to date (Ceredist) has been approved for the treatment of human spinocerebellar degeneration (Gary et al. 2003; Tanabe Seiyku Ltd 2002). Clinical and empirical evidence indicates a potential role for TRH in the treatment of depression, despite equivocal results of clinical trials possibly due in part to the short half-life of native TRH (Vetulani and Nalepa 2000; Sharma et al. 2001). Patients with bipolar II disorder may be especially responsive to the beneficial effects of TRH on mood (Marangell et al. 1997). Numerous actions of TRH have been identified as contributing to its capability to provide neuroprotection and improve recovery following CNS trauma and stroke. These include antagonism of the deleterious effects of several classes of secondary injury factors resulting from cell damage, such as endogenous opioids, peptidoleukotrienes, platelet activating factor and excitotoxins, as well as the capacity to improve blood flow, cellular bioenergetics and ionic homeostasis (Faden AI, Salzman 1992; Kelly 1995; Pitts et al. 1995). TRH has been shown to protect brain cells from glutamate-mediated neurotoxicity (Pizzi et al. 1999) and improve critical biochemical functions impaired by CNS trauma such as cell bioenergetics (Pitts et al. 1995), the art would suggest that it may offer important therapeutic advantages in this area. TRH's neurotrophic actions (Askanas et al. 1989) may be used for restoring loss of function associated with neurodegeneration or for preventing or retarding rates of further loss. At present, the mechanisms underlying the potential therapeutic actions of TRH are not fully understood. It is possible that the beneficial effects of TRH are multifactorial and could be due in part to its action in mitigating or reversing the secondary neuronal cell damage that is caused by a sequence of biochemical reactions triggered by the primary injury (Pitts et al. 1995). TRH has been shown to antagonise the actions of multiple constituents of the sequence leading to cell damage (Faden and Salzman 1992; Pitts et al. 1995). TRH has been shown to antagonise the effects of four classes of substances implicated in secondary tissue injury, including endogenous opioids, peptidoleukotrienes, platelet-activating factor and excitotoxins. This same sequence of reactions has been found to occur in both acute and chronic neurodegeneration and drugs capable of disrupting this sequence have potentially broad application as neuroprotectants (Jain PharmaBiotech Neuroprotection 2003). It is becoming evident that those targeting multiple components of the sequence may offer therapeutic advantages over pharmacological interventions targeted at single components (Callaway et al. 2004).

It has been established in the art that treatment with TRH is beneficial in cases of CNS trauma and other CNS disorders, for example, brain and spinal injury, epilepsy and spinocerebellar degeneration. In their paper examining TRH in the hippocampus of Alzheimer's Disease (AD) patients Luo et al note that the concentration of TRH was decreased in the hippocampus of AD patients compared to normal elderly controls and raise the possibility that TRH promotes a neuroprotective function so that a TRH deficiency may increase the vulnerability of such neurons to metabolic insult (Luo et al. 2002). Moreover, activation of TRH neuronal systems may be considered to provide a mechanism for tenninating excessive neuronal activity as is evident in epilepsy (Gary et al. 2003). Hence one therapeutic approach for such conditions would be to increase the bioavailability of TRH even transiently during and following injurious and/or disturbing events associated with physiological and pathological events inter alia loss of cognitive function linked to the normal aging process and neurodegenration, thereby in principle providing benefit subsequent to CNS injury and/or disturbances of neurobiological function (Kelly 1995) that can occur acutely, chronically or, for example, as a part of the aging process. Kelly, 1995, discusses the potential of TRH in the treatment of CNS disorders and states that the therapeutic utility of TRH is critically limited by its short half-life. Thus, strategies are needed to enhance the neuropharmacological efficacy of TRH in the treatment of such disorders as CNS trauma. Given a correlation between TRH levels and therapeutic outcome there is an unmet need to develop methods that increase TRH bioavailability and/or signalling. As stated in Kelly, 1995, a major problem undermining the therapeutic use of TRH is its rapid degradation in the CNS. This rapid degradation of TRH minimises the therapeutic efficacy of administered peptide. In human clinical trials TRH has been demonstrated to have a large therapeutic window and is well tolerated (Kubek and Garg 2002). The clinical utility of TRH is, however, severely limited by this susceptibility to enzymic degradation, which significantly reduces TRH bioavailability and duration of action (Kelly 1995). This is reflected in a disappointing performance produced by native TRH in clinical trials. The short half-life of TRH, arising from enzymic degradation, is also a serious impediment to further investigation of the functions of TRH. Approaches to address this problem have so far been directed at the development of degradation-stabilized TRH analogs and/or delivery systems such as microspheres, intranasal delivery and implanted microdisks, which transport additional TRH and/or TRH analogs to appropriate tissues. (Kelly 1995, US20020004062, US4906614, US5112804, US5244884, US5428006, US5686420, US5244884, US5693608, US5804212, US6303134, US6475989, US6491939, and US6524557).

US5244884 relates to thionated analogs of TRH type compounds, which selectively bind to TRH receptor binding sites in animals with high affinity and potentially have pharmacological advantages over TRH in treating those pathological conditions in which the effects of TRH have been shown to be beneficial. US5112804, US5428006 US5693608, US5804212, US6491939 and US6524557 describe methods for administrating a therapeutically effective amount of biologically active substances, including TRH.

US5686420 describes a series of novel TRH analogs wherein the C-terminal prolineamide moiety has been preserved, the N-terminal moiety comprises one of five different ring structures and the histidyl moiety is substituted with CF3, NO2 or a halogen and use of these analogs in the treatment of neurological disorders. US20020004062 describes methods and compositions for providing prolonged release of therapeutic agents, including TRH. Thus far, one degradation-stabilized analog has been approved for therapeutic use in humans; this was launched by Tanabe Seiyaku Co., Ltd., in 2000 under the trade name Ceredist and was assigned orphan drug status for the treatment of spinocerebellar degeneration (Tanabe Seiyku Ltd., 2002).

There therefore exists a problem presented by the short half-life of TRH. One solution proposed is to retard TRH degradation in CNS tissues (Kelly 1995; WiIk 1989; US20030166944). To date, the main focus has been on attempts to design analogues targeting TRH receptors, as classical enzymes inhibitors have been difficult to design due to the specificity of the enzyme The idea of using active site-directed TRH-DE inhibitors as pharmacological tools to potentiate the effects of exogenous TRH was discussed in a paper by WiIk in 1989 and subsequently by Kelly in 1995 and Bauer et al. 1997. All mechanisms to achieve this aim were focused on inhibition at the protein level - inhibiting enzyme. Although the development of inhibitors of the TRH-DE protein proved to be extremely difficult, J. A. Kelly discovered site-directed, competitive, reversible peptide TRH-DE inhibitors, which are disclosed in US20030166944. Simply, the present invention has not been proposed by any of these previous publications, which focus on the protein- the enzyme itself- as opposed to the process of expression of the gene encoding this enzyme, which is one of the subjects of this patent. With regard to TRH degradation, the key enzyme involved in degrading extracellular TRH is thyrotropin-releasing hormone-degrading ectoenzyme (TRH-DE) (EC 3.4.19.6), otherwise known as pyroglutamyl aminopeptidase II (PAP-II, PP-II). TRH-DE catalyzes the removal of the N-terminal pyroglutamyl group (GIp or pGlu) from TRH (WiIk 1989; Kelly 1995; Bauer et al. 1997) and is located on the surface of neuronal cells (Bauer et al. 1997). A soluble form of the enzyme, known as thyroliberinase, is present in serum (Bauer et al. 1997). The art suggests that TRH-DE has a close relationship with TRH in that TRH-DE does not appear to be responsible for the inactivation of other naturally occurring peptides that contain a N-terminal GIp residue. Further, TRH is not degraded by any other enzymes that are in a position to affect TRH signalling (Kelly 1995, Kelly et al. 2000). The two other enzymes known to degrade TRH (PAP-I and PE) are both cytosolic enzymes. These would not be expected to modulate extracellular TRH levels and thus far, the physiological significance of such enzymes is unclear (Kelly 1995). Inhibition of TRH-DE to make TRH more available and stable for delivery of potential therapeutic benefits has been explored at the protein level using peptidase inhibitors (WiIk 1989; Bauer et al. 1997; Kelly et al. 2000, 2005; US20030166944). In the art there is description of biological tools that enable targeted and sequence specific suppression of gene expression, protein expression and/or protein activity. One powerful technology is based on RNA enzymes, termed ribozymes, which can be designed to cleave target transcripts in a sequence-specific manner. Significantly, therapeutic ribozymes are in development for many genetic and infectious diseases (Millington-Ward et al. 2002, Tritz et al. 2005) or are at the stage of human clinical trials, for example, for disease targets such as HIV amongst others. In 1998 Andrew Fire and colleagues (Fire et al. 1998) described another tool for modulating or suppressing gene expression in C. elegans called interfering RNA (RNAi) or double stranded RNA (dsRNA). This tool was further refined for use in vertebrate tissues by using dsRNA molecules of 21-22 bp in length (termed short interfering RNA, or siRNA) (Elbashir et al. 2001). Potential applications for this biological tool are immense as RNAi has been shown to be effective in both mammalian cells and animals (Sandy et al. 2005). An important feature of dsRNA or siRNA or RNAi is the double stranded nature of the RNA and the absence of overhanging regions of single stranded RNA, although short overhangs of one, two or a few nucleotides may be tolerated. Parameters governing generation of functional siRNAs have not been folly established to date. Indeed, success for siRNA molecules is extremely variable, with some siRNAs not nearly as effective as other sequences.

Notably, specific suppression of target mRNAs with siRNA duplexes has now been demonstrated in mammalian cells and in vivo (Elbashir et al. 2001 ; Sandy et al. 2005) siRNAs may be delivered as synthesised RNA and/or using a vector to provide a supply of endogenously generated short hairpin RNAs which in turn can be processed into siRNAs (Sandy et al. 2005, Brummelkamp et al. 2002). Similarly, siRNAs, alone or in vectors, may be locally or systemically delivered.

In addition to siRNAs, microRNAs (miRNAs) represent a population of non-coding RNAs that are expressed in the cell. Recently, miRNAs have been used as a means of eliciting suppression of a target gene (Dickins et al. 2005, Zeng et al. 2005) and hence in a particular embodiment miRNAs may be used in the current invention.

A range of vehicles is available for delivery of nucleotide-based therapies including naked nucleotides, protected nucleotides and non-viral and viral vectors. Many of these vectors have been shown to effect delivery in cells, in animal models and in some cases in humans. Vectors include inter alia adenovirus, adenoassociated virus, retroviruses, lentiviruses, herpes virus, bacteriophage integrase systems, lipids and polymers, amongst others (Gardlik R et al.2005). Modifications to delivery vehicles can include inter alia chemicals or agents which aid in for example delivery across cell and/or nuclear membranes and/or which aid in tissue specific targeting of the therapeutic (Barnett et al. 2002; Kawano et al. 2004) and or in modulating cellular immune responses to vector. In addition, route(s) of administration can include local delivery, systemic delivery, intranasal administration (US5112804, US5804212, US5428006, US6524557) and delivery to one tissue that in turn aids delivery to additional tissue(s), for example, the use of nasal epithelia to deliver to brain tissues (US20030211966),) or intramuscular injection to deliver to spinal motor neurons (Azzouz et al. 2004). Object of the invention

One object of the invention is therefore to provide increased bioavailability of TRH especially for use in CNS disorders. A further object of the invention is to provide inhibitors of TRH-DE expression. Definitions

As used herein, the following terms have the given meanings unless expressly stated to the contrary.

The term 'bioavailability' may be defined as the degree to which a substance becomes available at the site of physiological activity.

The term 'increasing bioavailability', encompasses any means that acts to give a net increase on the biological effectiveness of the protein family, whether by means of increased expression, increased half life, altered binding efficiency, altered solubility, altered dissociation constants, increased reactivity or any combination of these or other commonly understood mechanisms of increasing the result of the relevant protein's actions.

An "oligonucleotide" is a nucleotide polymer having two or more nucleotide subunits covalently joined together. Oligonucleotides are generally about 10 to about 100 nucleotides in length, although they can be shorter, and may be longer, particularly in the case of antisense nucleotides of the invention, which may be hundreds or even thousands of bases in length. The term oligonucleotide is intended to encompass DNA, RNA, and DNA/RNA hybrid molecules. The sugar groups of the nucleotide subunits may be ribose, deoxyribose, or modified derivatives thereof such as OMe. The nucleotide subunits may be joined by linkages such as phosphodiester linkages, modified linkages or by non-nucleotide moieties that do not prevent hybridization of the oligonucleotide to its complementary target nucleotide sequence. Modified linkages include those in which a standard phosphodiester linkage is replaced with a different linkage, such as a phosphorothioate linkage, a methylphosphonate linkage, or a neutral peptide linkage. Nitrogenous base analogs also may be components of oligonucleotides in accordance with the invention. Oligonucleotides of the invention may be single stranded, double stranded, triple stranded, branched, comprise loops or take other structural forms or comprise additional moieties.

"Complementarity" is a property conferred by the base sequence of a single strand of DNA or RNA which may form a hybrid or double-stranded DNA:DNA, RNA:RNA or DNA:RNA through hydrogen bonding between Watson-Crick base pairs on the respective strands. Adenine (A) ordinarily complements thymine (T) or uracil (U), while guanine (G) ordinarily complements cytosine (C).

The phrase "substantially complimentary" means that the nucleotide has a nucleotide sequence substantially similar to a specified nucleotide sequence. Any additions or deletions are non- material variations of the specified nucleotide sequence which do not prevent the nucleotide from having its claimed property, such as being able to preferentially hybridize to or suppress TRH-DE RNA over other RNAs.Another definition which may be used is that by "substantially complementary" is meant nucleic acids having a sufficient amount of complementary nucleotides to form a hybrid complex and to effect supression.

By "substantially homologous" is meant nucleic acids having a sufficient amount of nucleotides identical to those nucleotides in a target sequence so as to be substantially complementary to the anti-sense or complementary strand of the target sequence.

The phrases "at least partially complimentary" and "at least partially homologous" refer to sequences that have at least a minimum degree of complimentartiy or homology to at least effect some increase in the bioavailability of TRH or TRH-DE-like peptide. This minimum degree is such that it is sufficient for the supresison agents of the invention to form at least partial and/or at least temporary interactions with the corresponding target sequence of TRH-DE so as to initiate some level of inhibition of TRH-DE translation, for example, by inducing cleavage and or degradation of transcripts amongst other mechanisms. Sequences of the invention are described as being partially homologous or partially complimentary, but it is understood that substantially complimentary or substantially homologous sequences may also be used. 100% complimentary or 100% homologous sequences may also be uised in most applications of the invention, except, for example, those situations where degeneracy is desired. Where the terms substantially complimentary or substantially homologous are used, it is understood that subsets of these groups (complimentary and homologous) are also included. In general, both terms are used, to account for the flexibility in ascribing sense and anti-sense strands, but where a particular application could not function with either a substantially homologous or substantiually complimentary sequence, then this is excluded respectively. "RNA and DNA equivalents" refer to RNA and DNA molecules having the same complementary base pair hybridization properties. RNA and DNA equivalents have different sugar groups (i.e., ribose versus deoxyribose), and may differ by the presence of uracil in RNA and thymine in DNA. The difference between RNA and DNA equivalents do not contribute to differences in substantially corresponding nucleic acid sequences because the equivalents have the same degree of complementarity to a particular sequence. Summary of the Invention

The present invention provides for a suppression agent for increasing the bioavailability of TRH comprising a nucleic acid comprising a supression sequence at least partially (may be substantially, and also may be 100%) complimentary to or at least partially (may be substantially, or may be 100%) homologous to a portion of TRH-DE RNA. Ideally, the portion of at least partially complimentary or at least partially homologous should be sufficient so as to be able to permit the supression sequence to bind to the sense or antisense sequence of the supression sequence on the TRH-DE RNA. The TRH-DE RNA may be mRNA. The TRH-DE RNA may comprise a sequence at least partially (may be substantially, or may be 100%) complementary to at least a portion of one or more of SEQ ID NO 11 , SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15 and SEQ ID NO 16.

Thus, the present invention provides agents and methods to increase the bioavailability, signalling and effects of TRH. The agents and methods of the invention provide surprising means of providing sustained bioavailability of TRH that act by inhibiting the bioavailability of TRH-DE. One of the advantages of the present invention over the present inhibitors, is that unlike inhibitors directed at the TRH-DE protein, nucleotide-based suppression can be engineered to be tissue specific.

Notably despite a wealth of literature on TRH and TRH-DE there has been no mention of suppression of TRH-DE expression in the manner provided for by the invention. The design and generation of suppression agents that result in either total absence of the TRH-DE protein or significant reductions in protein production represents a novel solution to the problem of TRH bioavailability due to short-half life and could provide significant advantages inter alia increases in longevity of TRH peptide. Targeting TRH-DE RNA and/or DNA provides the opportunity to generate suppression agents that operate either transiently and/or are produced by cell(s) and thereby provide a supply of suppressing agent(s) over a longer time frame. Additionally, an advantage of the invention is the ability to provide suppression agents in a tissue-specific and/or cell-specific manner. Moreover, suppression agents can be delivered in a form which is silent until 'induced' by agents inter alia chemical, light and heat utilising, for example, inducible promoters, thereby exerting control on dosage, timing and duration of the therapeutic agent.

While ribozymes and RNAi - represent powerful nucleic acid based tools for suppression of TRH-DE, the invention is not limited to the use of these agents. Many other suppression agents are available inter alia antisense RNA and DNA, nucleic acids, peptide nucleic acids (PNAs),triple helix DNA, miRNAs and others. The invention provides a means of efficient suppression of expression of TRH-DE targeting the TRH-DE gene (DNA) and/or transcripts from the TRH-DE gene (RNA). Suppression of TRH-DE expression can result in a reduction or absence of TRH-DE enzyme. In turn lowered levels of TRH-DE enzyme can result in increased levels of TRH peptide.

The invention provides for methods to silence expression of TRH-DE the key enzyme that limits the bioavailability of TRH. Nucleic acid-based suppression agents can be utilised to achieve silencing of TRH-DE inter alia, siRNA, ribozymes, DNAzymes, dsRNA, antisense, miRNA, triple helix. Suppression agents may be delivered alone or in combination. Nucleic acid-based suppression agents may be delivered alone or with compounds to aid cellular delivery inter alia lipids and/or polymers. Nucleic acid-based suppression agents may delivered alone or be incorporated into viral and/or non-viral vectors to aid delivery and longevity of suppression.

The supression sequence may be about 8 bases to about 100 bases in length. In some embodiments of the invention, the supression sequence is about 15 to about 27 bases in length. These values represent the preferred size of an siRNA molecule. siRNA molecules are typically composed of a double stranded molecule of RNA. In some embodiments of the invention, the supression sequence comprises an siRNA molecule. siRNA molecules may have short, single stranded overhangs. While these may be any base, it is preferred that they are homologous to the corresponding base in TRH-DE RNA. siRNAs or RNAi can be designed to target transcripts from a gene in a sequence specific manner. RNAi may be synthesised and/or may be expressed from one or more vectors; the latter are typically referred to as small hairpin RNAs or shRNAs (Brummelkamp et al. 2002). siRNAs have been used in cells and in animals and have elicited efficient and sequence specific suppression of target genes (Elbashir et al. 2001, Sandy et al. 2005).

MicroRNAs (nu^'RNAs) represent a family of endogenously produced RNAs which are believed to be involved in fundamental biological processes such as development, cell cycle control amongst others. miRNAs can be engineered to elicit sequence specific suppression of expression of a target gene in a somewhat similar manner to the suppression obtained with siRNAs (Dickins et al. 2005, Zeng et al.2005). The use of artificially produced miRNAs to elicit gene silencing has been proposed by Zeng et al. 2005 amongst others. Thus, vectors, plasmids or other nucleic acid based systems may be designed to provide for in vivo expression of the molecules of the invention, thus producing miRNAs. A suitable miRNA expression system is one as described in Dickins et al. 2005 or Zeng et al. 2005, albeit using TRH-DE RNA and/or the sequences of the present invention as appropriate to permit expression of an miRNA molecule of the invention.

MicoRNAs are small noncoding RNAs that are expressed in the tissues of many eukaryotes. MiRNAs are expressed as part of a transcript termed pri-miRNA embedded in which is a structure of approximately 80 nucleotides termed the pre-miRNA. The pre-miRNA in turn is processed to form approximately 20 base pair RNA duplexes typically with 2 nucleotide 3' overhangs. It is believed that these sequences are involved in controlling many fundamental cellular processes. The majority if not all miRNAs are believed to be expressed using RNA polymerase II for transcription. Notably, the majority of mammalian genes are indeed also expressed using RNA polymerase II, that is, mammalian genes typically have what is termed polymerase II promoter sequences. The present invention provides for artificial microRNAs comprising RNA PoIII and or RNA PoIIII promoter sequence(s). Thus, the pri-miRNA and/or pre-miRNA may be processed intracellularly such that small double stranded RNAs are generated which are complimentary or partially complimentary to the target gene or transcript of interest. Such a system enables ready incorporation of polymerase II promoters in such constructs facilitating the generation of a system enabling tissue specific suppression of gene expression. The advantages of using miRNAs provided by the invention include the ability to inhibiting TRH-DE gene expression in a tissue specific manner.

Indeed, the invention also provides for tissue specific supresionn agents. It is notable that regardless of the suppression agent utilised inter alia, siRNA, miRNA, ribozymes, antisense, there are potential advantages to tissue specific suppression of TRH-DE gene expression including but not exclusive to increasing the resolution of a research tool or minimising side effects associated with a therapy. In this regard any of the suppression agents in the invention can be expressed using ubiquitous and/or tissue specific and/or inducible promoters to control the expression of the suppression agent in terms of tissue or cell type, location, level of expression or timing of expression. The suppression agents of the invention also comprise means to permit tissue specific expression as detailed in PCT/GB2003/00381. The siRNA molecules of the invention (and miRNA molecules of the invention) can be designed to be at least partially (may be substantially, or may be 100%) homologous to or at least partially (may be substantially, or may be 100%) complimentary to at least a portion of TRH-DE RNA, and/or to one or more of SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15 and SEQ ID NO 16. The siRNA molecules (and miRNA molecules) of the invention may comprise a supression sequence at least partially (may be substantially, or may be 100%) homologous to or at least partially (may be substantially, or may be 100%) complimentary to SEQ ID NO 1. Or SEQ ID NO 2. Or SEQ ID NO 3. Or SEQ ID NO 4. Or SEQ ID NO 5. Or SEQ ID NO 6. Or SEQ ID NO 7 or the sequences disclosed in Table 2. The nucleic acids of the invention may also be delivered as single stranded nucleic acid sequences. Such molecules would then be antisense molecules. Antisense molecules may be of any suitable length, and may be between about 8 and about 100 bases in length, (may be also between about 8 and about 50 bases) but may be hundereds or even thousands of bases in length. For example, antisense molecules of the invention may comprise an anti-sense molecule directed to all or a substantial length of the TRH-DE RNA sequence.

Nucleic acids encoding suppression agents inter alia RNAi and/or ribozymes and/or miRNAs for suppression of gene expression may be provided in the same vector and/or in separate vectors. Suppression agents such as RNAi can be delivered as naked DNA, modified DNA, naked RNA, modified RNA and/or in a carrier vehicle(s) or vector(s). Nucleotide modifications can be made to improve efficacy and/or longevity of suppression agents and/or to reduce cellular toxicity associated with suppression agents, for example, by evading the immune system. Naked nucleic acids or nucleic acids in vectors can be delivered with lipids and/or other derivatives which aid delivery and/or reduce toxicity. Nucleotides may be modified to render them more stable, for example, resistant to cellular nucleases and/or with increased binding efficiencies.

Complete silencing / suppression of a gene or allele or RNA in some instances may be difficult to achieve using suppression agents inter alia ribozymes and/or RNAi. However, a reduction in quantities of gene product may provide beneficial effect(s). Such a reduction in TRH-DE may result in increased levels of endogenous TRH peptide and/or administered TRH and/or TRH analogues.

Use of inhibitors or silencers such as ribozymes, and/or siRNA and/or antisense and/or miRNA directed towards a specific TRH-DE variant or splice variant can permit a high degree of specificity in targeting both expression levels and tissue specific locations. In a particular embodiment, one or more splice variants of the target gene may be suppressed using suppression agents. In another embodiment suppression of TRH-DE expression is directed to one or more tissues.

The suppression agent may comprise an antisense nucleotide or an siRNA molecule. The suppression agent of the invention may comprise an oligonucleotide comprising a sequence at least partially (may be substantially, or may be 100%) complimentary to or at least partially

(may be substantially, or may be 100%) homologous to one or more of SEQ ID NOs 1 -7.

In some embodiments of the invention, the suppression agent comprises an antisense or siRNA molecule or or a miRNA selected to be at least partially (may be substantially, or may be 100%) complimentary or substantially complimentary in sequence to at least a portion of the RNA of

TRH-DE.

In additional aspects of the invention, the agents of the invention comprise a ribozyme. In such embodiments the supression sequence is up to about 100 bases in length, and is preferably up to about 70 bases in length, and more preferably about 15 bases to about 45 bases in length.

Ideally, the ribozymes of the invention comprise a base catalytic core of about 25 bases and may further comprise one or two arms of about 3 to about 16 bases. T he ribozyme of the invention may be at least partially (may be substantially, or may be 100%) complimentary to or substantially homologous to SEQ ID NO 10. Further ribozymes of the invention are also envisigned, provided they are designed according to the requirements as described herein and are at least partially homologous or partially complimentary to TRH-DE RNA, and/or portions of SEQ ID NOs 11-16.

In further aspects of the invention, the suppression agents of the invention comprise miRNAs which are complementary or at least partially (may be substantially, or may be 100%) complementary to at least a portion of TRH-DE RNA, in particular, one or more of SEQ ID NO

1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7,

SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO

16. In some embodiments suppression effectors can be synthesised exogenously and then administered to the target cell(s), tissue(s), organ(s) and/or animals and/or can be generated within cell(s) (endogenously) from one or more vehicles or DNA fragments in the cell(s). In further embodiments, suppression agents targeting TRH-DE DNA and/or RNA are provided in combination with one or more agents targeting the TRH-DE protein, for example, small peptide inhibitors of the TRH-DE enzyme, such as those described US patent application number: 0030166944. In further embodiments suppression agents targeting TRH-DE DNA and/or RNA are provided in combination with one or more inhibitors or agents that may act to protect TRH from degradation for example inter alia, inhibitors of TRH-DE (EC 3.4.19.6), otherwise known as pyroglutamyl aminopeptidase II, pyroglutamyl aminopeptidase I (EC 3.4.19.3) and/or the prolyl oligopeptidase (EC 3.4.21.26) (PE). Further suitable examples of peptide and protein inhibitors of TRH-DE are described in PCT/IEO 1/00027; US0030166944, and Kelly A, Scalabrino GA, Slator GR, Cullen AA, Gilmer JF, Lloyd DG, Bennett GW, Bauer K, Tipton KF, Williams CH. Structure-activity studies with high-affinity inhibitors of pyroglutamyl-peptidase II. Biochem J. 2005 JuI 15;389(Pt 2):569-76. Preferred TRH-DE inhibitors include Glp-Asn-Pro-Tyr-Trp-Trp-7-amido-4methylcoumarin with a Ki of 1 nM. Other favourable TRH-DE inhibitors are Glp-Asn-ProNH₂ and Glp-Asn-ProAMC with Ki values of 14 μM and 1 μM, respectively.

The agents of the invention may be delivered systemically and/or may be locally delivered and/or may be delivered to a local site for subsequent transport to an alternative site to the target cell(s), tissue(s) and/or organ(s) and/or animal(s). Suppression targeting TRH-DE at the RNA and/or DNA level(s) may be undertaken in test tubes, in cells, in tissues, in organs and/or in animals.

The invention further provides for suppression effectors(s) targeting TRH-DE at the RNA and/or DNA level(s) are designed in a carrier such that a supply of the suppression agent(s) is provided which has a longer time-frame and or bioavailability than if the suppression agent is provided alone, for example, such as described in (Bonsted et al. 2006). The invention also provides for suppression agents further comprising a vector {inter alia, expression vectors) for delivering the suppression agent to cells, tissues or physiological locations. The vector may comprise suppression agents inter alia ribozymes and/or RNAi and/or antisense and/or miRNA sequences capable of interfering with target TRH-DE RNA(s) and/or DNA(s). The vector may be inter alia viral, non-viral, naked DNA and/or RNA and/or modified nucleotides, artificial chromosomes or any vehicle for delivery of the suppression agents (Gardlik R et al. 2005). Exemplary viral vectors that may be used in the practice of the invention include those derived from adenovirus; adenoassociated virus; retroviral-C type such as MLV; lentivirus such as HIV or SIV; herpes simplex (HSV); and SV40. An exemplary non- viral vector includes the Streptomyces bacteriophage phiC31 integrase (Ortiz-Urda et al. 2002). Cationic lipid mediated delivery of suppression effectors, soluble biodegradable polymer-based delivery, or electroporation/ iontophoresis may also be used amongst other methods. In another aspect, the invention provides vectors (inter alia, expression vectors) comprising nucleotide sequences encoding one or more suppression agents, for example, RNAi and/or ribozyme(s) and/or suppression agent(s) capable of interfering with a TRH-DE RNA and/or DNA. The vectors of the invention may be viral, non-viral, naked DNA and/or RNA and/or protein, artificial chromosomes or any vehicle for delivery of suppression agent(s). In an embodiment, the suppression agents, for example, the RNAi encoding sequence(s) and the ribozyme sequence(s) are in the same vector. In an alternative embodiment, the suppression agents, for example, the RNAi encoding sequence(s) and ribozyme sequence(s) are present in different vectors.

In a further embodiment of the invention expression of suppression agents in a vector(s) is controlled by the tissue specificity of the promoter(s) that drive expression of suppression agent(s). In an additional embodiment inducible expression of suppression agents is described. The invention provides for control of the timing, dosage and/or duration of expression of suppression agents targeting TRH-DE. The suppression agent may be administered in combination with TRH or TRH-like peptide or a nucleotide encoding TRH or TRH-like peptide. The suppression and/or TRH and/or TRH-like peptides may be delivered by a vector to cells, tissues or any physiological locations.

The invention may be applied inter alia in the development of therapeutics for any TRH-related disorders, inter alia, spinal injury, memory loss, spinocerebellar degeneration, spinal cord pain, epilepsy, obesity, diabetes, psychiatric disorders, disorders of mood and CNS related diseases and as a research tool to investigate TRH and TRH-DE related cellular processes and in the generation of transgenic cells and animals

The invention also conceives of the use of a suppression agent as herein described in the preparation of a medicament, or in the manufacture of a pharmaceutical composition, ideally to be used for the treatment of a disorder of the CNS, especially any of the disorders of the CNS as discussed herein. A pharmaceutical composition may also further comprise a suitable pharmaceutical carrier.

TRH-DE suppression hence may be used, for example, in the treatment of TRH-related disorders. This therapeutic approach may be augmented by the suppression of additional targets, for example, the TRH degrading enzyme, prolyl oligopeptidase. Suppression agents may be delivered alone and/or with a compound to augment therapeutic efficacy such as exogeneous TRH. Suppression agents may also be delivered with compounds to aid infection, transduction and/or transfection of target cells.

The compounds described in the present invention may be administered by inter alia oral, parenteral, intramuscular (i.m.), intraperitioneal (i.p.)₅ intravenous (i.v.) or subcutaneous (s.c.) injection, and inter alia nasal, vaginal, rectal or sublingual routes of administration and can be formulated in dosage forms appropriate for each route of administration. Suitable dosage forms are known to those skilled in the art and are described, for example in US4906614 Giertz et al. or US5244884 Spatola et al.

Desirably, the invention provides for use of a compound and/or vector in a pharmaceutically acceptable salt thereof in the preparation of a medicament, particularly for the treatment of brain or spinal injuries or other central nervous system disorders or other TRH dependent disorders. Desirably also, the invention provides a method of treatment of brain or spinal injuries or other central nervous system disorders or other TRH-dependent disorders, which comprises administering to a patient suffering from such injuries or disorders an amount of a compound and/or vector in a pharmaceutically acceptable salt thereof effective to potentiate endogenous TRH and /or protect exogenously administered TRH or TRH analogues from degradation by TRH-DE and/or act at TRH receptors.

The invention also provides a method of treating a disorder of the CNS comprising administering a suppression agent or a modulating agent as herein described to a human and/or non-human patient.

The term "treat" as used herein is intended to encompass prophylaxis as well as curing and/or ameliorating at least one symptom of the condition or disease. Likewise, a "therapeutic" is an agent which provides prophylaxis against or cures or ameliorates at least one symptom of the condition or disease. The invention may be applied in therapy approaches for biologically important disorders affecting certain cell types or cell subpopulations. Similarly the approach may be used to modulate normal and/or dysfunctional physiological processes. The invention may also be used in the investigation of the biological mechanisms and cellular processes related to the target TRH-DE and substrates of the target, and/or related to the target TRH receptor(s) and ligands thereof, for example, TRH. A further use of the invention is the generation of transgenic cell lines and/or animals with reduced levels of the TRH-DE enzyme. The invention also provides for methods of testing suitable suppresison agents of the invention comprising selecting suppression agents that are equally homologous or complimentary between human THR-DE (preferably SEQ ID No 11 or SEQ ID NO 12) and one or both of mouse TRH- DE (preferably SEQ ID No 11 or SEQ ID NO 12) and rat TRH -DE (preferably SEQ ID No 11 or SEQ ID NO 12). Such suppression agents permit the same sequence to be tested in a laboratory animal (mouse or rat respectively) and then moved to clinical trials in humans. Figure 7 details a sequence alignment of SEQ ID NOs 11, 13 and 15. siRNA molecules and miRNA molecules of the invention that would be suitable for such cross-species studies would be apparent as those that have a high to 100% degree of complimentarity across the desired species for the defined or preferred suppression sequence length.. In addition, while not wishing to be bound by theory, it is believed that portions of the sequence that show a high to 100% homology across species are likely to be particularly important sequences with respect to functionalilty of the encoded TRH-DE protein. Thus, advantageous siRNA molecules, miRNA molecules, antisense nucleotides and/or ribozymes of the invention may be directed toward these high to 100% homologous regions, so as to have a greater likelihood of being effective against all major and minor variants and/or splice variants of the TRH-DE protein.

Brief Description of the Drawings

Figure 1. Graphical representation of hTRHDE down regulation by TRH-DE specific siRNA molecules using GAPDH as a standard. Figure 2. Graphical representation of hTRHDE down regulation by TRH-DE specific siRNA molecules using Neomycin as a standard. Figure 3. Graphical representation of pIRES2-EGFP-hTRHDE expression in the presence of various siRNA molecules. Figure 4. Graphical representation of rTRHDE down regulation by TRH-DE specific siRNA molecules using GAPDH as a standard. Figure 5. Graphical representation of rTRHDE down regulation by TRH-DE specific siRNA molecules using Neomycin resistance gene as a standard. Figure 6. RzTRH-DEs designs and predicted 2-D structure of the target human TRH-DE transcript.

Figure 7:. Clustal Sequence Alignment of SEQ ID NOs 11, 13 and 15.

Examples

EXAMPLE 1 : siRNA targeting hTRH-DE siRNA(s) targeting any region of the TRH-DE sequence can be used in the invention — some examples are provided in Table I. Experiments evaluating down-regulation of a TRH-DE target have been undertaken by co-transfecting COS-7 cells with siRNA(s) targeting TRH-DE and the TRH-DE pIRES2-EGFP plasmid or the pCDNA3.1 plasmid (pc3. IhTRHs) using art known methods. pIRES2-EGFP enables expression of the target transcript together with enhanced green fluorescent protein (EGFP) transcript. Stable cell lines expressing the TRH-DE-EGFP construct can be generated using standard art known methods and further suppression experiments can be carried out in stable cell lines. For example, stable lines can be transiently transfected with TRH-DE specific RNAi molecules (siRNAs). Suppression of the TRH-DE target by siRNAs can be assessed using art known methods inter alia real-time (rt) PCR, northern blotting, fluorometric evaluation of levels of GFP protein and western blotting. While COS-7 cells and RNAi have been used as examples, other cell lines and other suppression agents may be used in the invention.

Table 1 : siRNA sequences

Table 1 presents information on SEQ ID NO, siRNA title and the target sequence in TRH-DE targetted by suppression agents. It is to be understood that the antisense version of SEQ ID Nos 1-7 are appropriate to use as single stranded antisense molecules, and double stranded versions are applied where siRNA, miRNA are used.

Similarly, Table 2 shows further siRNA molecules which are suitable for use in the invention. Table 2 : hTRH-DE siRNAs

Human TRH-DE (hTRH-DE) cDNA has been cloned into plasmid pCDNA3.1 (Clontech). (pc3.1hTRHse) 2μg of purified hTRHDE plasmid was transiently transfected into pre-seeded Cos7 cells, in a 6 well format, by means of Lipofectamine 2000 (Invitrogen) transfection reagent. RNA was extracted, purified and analysed by real time RT-PCR according to standard protocols. Maximal expression of the hTRHDE plasmid, under the stipulated transfection conditions and concentration, was found to occur 24 hours post-transfection and declined gradually subsequent to this time point. siRNA-based suppression of TRH-DE was assessed at 30 hours post transient transfections of the target gene and siRNA in COS-7 cells. Figures 1 and 2 depict the suppression efficiency of siRNAs targeting human TRH-DE (hTRH-DE). More specifically, 2μg of hTRHDE plasmid was transiently transfected into Cos7 cells, in conjunction with Qiagen™ synthesized TRHDE-targeting siRNA, as specified in the manufacturer's protocol (transfection reagent used was Lipofectamine™ 2000, Invitrogen™). 40pmoles of each siRNA molecule was added to respective cells, in addition to 2μg of hTRHDE plasmid. Cell cultures were incubated at 37⁰C and 5.1%CO₂ for 30 hours prior to RNA extraction. The relative levels of hTRHDE mRNA in isolated RNA samples was analysed by RT-PCR using GAPDH as the housekeeping gene. As depicted in figure 1, siRNA-based suppression is expressed relative to a control non-silencing / non-targeting siRNA. This value is assumed to represent maximal expression of hTRHDE (and thus designated a value of 100) and levels of siRNA-based suppression are determined or normalised to this value, yielding the relative expression values presented in the figure (Figure 1). I)TRHDE₄₄₃SiRNA and hTRHDEi₄o₇siRNA suppressed expression of hTRHDE by 66.09±18.04 %(p<0.01) and 68.12±15.61% (pO.OOl) respectively compared to the control siRNA. H&MsiRNA and H&M&RsiRNA specifically down-regulated hTRHDE expression by 63.32±18.13% (p<0.01) and 61.85+16.11% (p<0.01) respectively, again relative to the control siRNA. In a manner similar to that described above, Cos7 cells were transiently transfected with hTRHDE plasmid. RNA was extracted 24 hours post transfection and real time RT PCR analysis was carried out using Neomcyin as a marker for transfection efficiency and plasmid expression. Figure 2 depicts siRNA-based suppression of hTRH-DE expressed relative to a control non-silencing / non-targeting siRNA. This value is assumed to represent maximal expression of hTRHDE (and thus designated a value of 100) in the transiently transfected Cos7 cells; all remaining calculations are normalised to this value yielding the relative expression values presented in Figure 2. MTRHDE₄₄₃SiRNA and JiTRHDEi₄₀₇SiRNA suppressed hTRHDE by 72.5+11.98% (p<0.01) and 69.09±12.54% (p<0.001) respectively compared to the control siRNA. H&MsiRNA and H&M&RsiRNA specifically down-regulated hTRHDE expression by 74.38+10.02% (pO.OOl) and 78.12+7.73% (p<0.001) respectively, again relative to the control siRNA.

Cos7 cells were transiently co-transfected with varying siRNAs and a plasmid expressing the human TRHDE target (pIRES2-EGFP-hTRHDE). Note in this case the hTRHDE and EGFP sequences are expressed as part of a single RNA transcript using this expression plasmid. Each transfection was undertaken in triplicate and RNA samples extracted 48 hours post-transfection, pooled and evaluated by real time RT PCR. The mean percentage suppression from two independent experiments of this nature was determined. Col7Al siRNA acted as a non-targeting siRNA, and thus was designated a numerical value of 100 with respect to maximal hTRHDE expression (Figure 3). All other data was normalized to this value. Qiagen™synthesized 'TRHDE siRNA' suppressed TRHDE expression by 11.3%+0.5 while the initial 'batch' of T7 in vitro transcribed siRNAs yielded efficient suppression of the target (Figure 3 - labelled 'old' in the legend). hTRHDE₄₄₃ siRNA suppressed mRNA levels by 75%+0.05, hTRHDE₆₅₇siRNA down regulated hTRHDE by 70%+1.2 while hTRHDE₁₄₀₇siRNA suppressed hTRHDE by 76%+0.7. As a positive control siRNA targeting EGFP was found to down regulate TRHDE expression by 66%+4.2 and in vitro transcribed GFPsiRNA suppressed EGFP (and hTRHDE) expression by 75%+0.1. The second batch of in vitro transcribed siRNAs (labelled 'new' in the legend) resulted in a lower potency with respect to suppression of TRHDE gene expression. hTRHDE₄₄₃siRNA, hTRHDE₆₅₇siRNA, hTRHDE₁₄₀₇siRNA and in vitro transcribed GFPsiRNA suppressed TRHDE by 74%+1.0, 42%+3.8, 46%+2.5 and 42%+2.4 respectively. EXAMPLE 2

Similar to the protocol carried out for the hTRHDE plasmid; rat TRHDE (rTRHDE) (2μg) was transiently transfected into Cos7 cells. RNA was extracted at 12 hour intervals and analysed by real time RT PCR. rTRHDE plasmid expresses optimally 24 hours post-transfection, declining significantly subsequent to this time point. Thus RNA samples for suppression analyses were extracted 24 hours post-transfection.

In accordance with the standard protocols known in the art, for example, Elbashir et al. 2001, 2μg of the rTRHDE plasmid was transiently transfected into Cos7 cells, in conjunction TRHDE-targeting siRNA, using Lipofectamine 2000 (Invitrogen). 40pmoles of each siRNA molecule was added to cells in addition to the target (rTRHDE) plasmid. Cell cultures were incubated at 37⁰C and 5.1%CO₂ for 24 hours prior to RNA extraction. The relative expression levels of rat TRHDE was analysed by RT-PCR using GAPDH as a housekeepng gene. Figure 4 depicts siRNA-bsaed suppression of rTRHDE expression expressed relative to a non-silencing / non-targeting control siRNA. hTRHDE₄₄₃siRNA transfected cells expressed rTRHDE to a value of 35.79+12.49% (p<0.05) relative to the non-targeting control siRNA suggesting that hTRHDE443siRNA suppressed the target gene to a value of 64.21+12.49% (p<0.05). In contrast, hTRHDEi₄o₇siRNA only down-regulated the gene by 7.93+9.79% (p<0.05) relative to the control siRNA. H&MsiRNA and H&M&RsiRNA specifically down-regulated rTRHDE expression by 64.93+1.92% (p<0.001) and 82.75+4.02% (p<0.001) respectively again relative to the non-targeting control siRNA. In addition, HiTRHDE₇₃₄SiRNA suppressed expression of the target gene, rTRHDE by 22.3±21.34% (p<0.05) compared to the non-targeting siRNA control. (Check labelling for HiTRHDE₇₃₄SiRNA on Figures 4 and 5) rTRHDE plasmid was transiently co-transfected with various siRNA molecules into Cos7 cells and real time RT PCR analysis of isolated RNA carried out using primers targeting the Neomycin resistance gene. Figure 5 depicts siRNA-based suppression of rTRHDE expression (relative to a control non-targeting siRNA). The value for the non-targeting control is assumed to represent maximal expression of the rat TRH-DE gene (and thus has been designated a value of 100) in the transiently transfected Cos7 cells. hTRHDE₄₄₃siRNA suppressed rTRHDE to a value of 70.81+12.26% (p<0.05) relative to the non-targeting control siRNA. H&MsiRNA and H&M&RsiRNA specifically down-regulated rTRHDE expression by 63.93+9.39% (p<0.05) and 85.19+2.8% (p<0.01) respectively (again relative to the non-targeting control siRNA). siRNAs developed as exemplified above may be delivered as synthesised nucleotides and/or in viral and/or non- viral vectors (or as plasmids or DNA fragments) from which shRNAs are expressed within transfected cells. Synthesised siRNAs or vectors expressing shRNAs may be administered to animals and the down-regulation of TRH-DE evaluated using inter alia realtime rt PCR, northern blotting, western blotting, immuno cytochemistry, ELISA assays. Additionally, behavioural features associated with increased levels of TRH may also be evaluated (see Example 3 for details of behavioural read-outs). TRH-DE suppression agents can be administered into animals via inter alia tail vein injection, injection into the lateral cerebral ventricles and/or intrathecal injection using art know methods. Therapeutic TRH-DE suppression agents can be administered systemically and/or locally for example, to the CNS. The physiological readouts for in vivo suppression referred to above can be used to analyse animal behaviour patterns subsequent to administration of suppression agents. EXAMPLE 3 : Ribozymes

In some embodiments the suppression agents of the invention comprise one or more ribozymes targeting TRH-DE mRNA. Ribozymes of the invention can be designed to cleave an RNA molecule using specific ribozyme arms which bind to a particular RNA on either side 5' and/or 3' of a consensus sequence, for example, that described by Hasselhoff and Gerlach, 1992, Thus, any RNA sequence possessing consensus sequence sites represents a potential target. Notably, different classes of ribozymes have different sequence requirements for cleavage. Moreover, other variables require consideration in designing ribozymes, such as the two dimensional conformation(s) of the RNA (e.g., loop structures) and the accessibility of a ribozyme for its target. The utility of an individual ribozyme designed to target a consensus site in an open loop structure of a transcript will depend in part on the robust nature of the open loop structure which may be predicted using an RNA-folding computer program. Robustness of loop structures can be predicted over a broad range of energy profiles according to art known parameters. The ribozymes of the present invention include inter alia hairpin, hammerhead, trans-splicing ribozymes. In addition, any RNA inactivating or RNA cleaving agent which is capable of recognition of and/or binding to specific nucleotide sequences in an RNA (e.g. splicesome- mediated RNA trans-splicing) is contemplated. Ribozymes can be composed of modified oligonucleotides (e.g., for improved stability, targeting, etc.). A preferred method of delivery involves using a DNA construct "encoding" the ribozyme under the control of a ubiquitous, tissue specific, cell specific and/or inducible promoter, so that transfected cells will produce sufficient quantities of the ribozyme(s) to cleave the RNA target.

The sequences in figure 6 represent a ribozyme that has been designed using PlotFold to predict open loop structures in the transcript. The antisense arms of the ribozyme are designed such that they are complimentary to the TRH-DE RNA. The hammerhead ribozyme presented utilises a consensus sequence described by Hasseloff and Gerlach 1992. This consensus sequence is flanked by ribozyme antisense arms which are complimentary to the target RNA. The antisense arms of the ribozyme are underlined. It is notable that for a hammerhead ribozyme to cleave the target transcript, an NUX site is required where N is any nucleotide, U is Uridine and X is any nucleotide except G.

In an additional example, detailed RNAPlotFold analyses for human and rat TRH-DE transcripts have been undertaken and potential hammerhead ribozyme cleavage sites (NUX sites) in robust open loop structures of target transcripts identified (Figure 6). While Figure 6 provides details of a hammerhead ribozyme that can be evaluated for cleavage of TRH-DE target transcripts in principle ribozymes can be targeted to any area of the TRH-DE transcript. DNA primers for TRH-DE ribozymes (RzTRH-DEs) can be synthesised commercially and cloned into a vector such as pcDNA3.1 (Invitrogen) using art known methods. pCDNA3.1 enables expression from the T7 promoter in the vector. RzTRH-DE can be in vitro transcribed using Ribomax kits (Promega). Human and rat TRH-DE cDNAs can also be cloned into pcDNA3.1 or a similar vector. Target transcripts can be expressed in vitro from the T7 promoter in the vector incorporating P32dUTP to enable autoradiographic visualisation of target transcripts and ribozyme cleavage products. Ribozymes follow Michaelis-Menten kinetics (Fedor and Uhlenbeck 1992; Stage-Zimmerman and Uhlenbeck 1998). Detailed kinetic profiles in vitj-o can be established for RzTRH-DEs to determine their in vitro efficiencies using standard art known methods. Kinetic parameters such as the maximum velocity of cleavage (Vmax), ribozyme association and dissociation rates (kl and k-1) and the rate of the actual cleavage step (k2) can be determined to provide estimates of ribozyme cleavage efficiencies in vitro using art known protocols (Millington-Ward et al. 1999; O'Neill et al. 2000). In one aspect of the invention ribozymes are utilised alone or in combination with one or more suppressing agents to elicit sequence specific suppression of the TRH-DE gene by targeting RNA transcripts from this gene. A partial or complete reduction of TRH-DE RNA levels can result in a reduction in TRH-DE protein levels. Reduction in TRH-DE protein levels may increase the longevity and/or efficacy of TRH peptide. The invention provides for RNAi used alone or in combination with one or more suppression agents to elicit suppression of TRH-DE expression. In a further embodiment of the invention multiple suppression effectors inter alia RNAi, ribozymes, antisense, PNAs, triple helix, miRNAs are used together or alone to suppress expression of the TRH-DE target.

Subsequent to design, generation and in vitro kinetic evaluation of RzTRH-DEs, ribozymes determined to be efficient in vitro can be assayed for activity in cell culture. Stable cell lines expressing human and/or rat TRH-DE in pIRES2-EGFP or alternatively cells transiently transfected with the target genes can be used both for studies with RNAi and ribozymes. Transient transfection of RzTRH-DEs ribozyme constructs can be undertaken in cells using a transfection agent such as lipofectamine plus or oligofectamine which have been optimised for transfections. The ability of ribozymes to target and suppress TRH-DE can be evaluated using inter alia real-time PCR, rt-PCR, northern blotting, western blotting, fluorometric analyses and light microscopy on fixed cells using art known methods. Additionally double stable cell lines expressing human TRH-DE and therapeutic ribozyme(s) can be generated (using a second antibiotic resistance marker for selection of the presence of ribozyme construct(s) in stable cell lines).

Possible methods of delivery of therapeutic ribozymes and RNAi are described in the art. Ribozymes can be synthesised and delivered as naked or modified nucleotides (Goodchild 2002). Additionally viral and non-viral methods of delivery can be used (Aigner et al. 2002). Notably, a number of viruses have been used to achieve gene transfer into the CNS (Janson et al. 2002; Passini et al. 2001). One such virus is adenoassociated virus (AAV). Generation of AAV vector(s) with ribozymes and/or RNAi targeting TRH-DE can be undertaken using art known methods. Recombinant virus can be used to explore suppression of the target TRH-DE in cells and in vivo. The art describes delivery of a variety of suppression agents both in viral and non-viral vectors and both to cells and to animals (using a variety of routes of administration). For example, recently a recombinant retrovirus carrying hammerhead ribozymes targeting human collagen IAl has been generated and has resulted in down-regulation of the Collagen IAl target in human bone stem cells (Millington-Ward et al. 2002).

TRH-DE gene suppression can be evaluated in vivo and potential therapeutic benefits assessed. In this regard it is notable that rat has been used previously as a clear readout for increased TRH activity in vivo (Kelly et al. 2000) and hence can be used as a marker for inter alia ribozyme- based and/or RNAi-based TRH-DE inhibition. A variety of behavioural effects are indicative of increased TRH availability in the rat CNS. The best recognised of these are increased locomotor activity, forepaw tremor and 'wet dog shakes' (WDS) (Webster et al, 1982, Yamada et al., 1984). However, a wide range of additional markers may be used to assess efficacy of suppression, including inter alia protection from CNS trauma in rat injury models (Faden et al., 1999, Lauer et al., 2000, Mclntosh et al 1988 Faden et al 1989), histological and neurological measurements (Baykal et al, 1996, Ceyaln et al, 1990 Akedemir et al, 1993, Pitts et al., 1995). The amplitude of these effects in animals can be used as an indicator of effective in vivo suppression using inter alia RzTRH-DEs and/or RNAi-TRH-DE. Furthermore, ribozymes and/or RNAi can be designed such that RzTRH-DEs ribozymes and/or RNAi-TRH-DE will target both human and rat TRH-DE transcripts - therefore notably TRH-DE ribozymes and/or RNAi that would potentially be therapeutically of value to humans can be evaluated in rats potentially circumventing the need to generate transgenic animals expressing the human TRH- DE gene. Notably, stable cells lines expressing rat TRH-DE are available to ensure that human TRH-DE ribozymes and RNAi are active against rat TRH-DE transcripts. TRH-DE suppression agents can be administered into animals via inter alia tail vein injection, injection into the lateral cerebral ventricles and/or intrathecal injection using art know methods. Therapeutic TRH-DE suppression agents can be administered systemically and/or locally for example, to the CNS. The physiological readouts for in vivo suppression referred to above can be used to analyse animal behaviour patterns subsequent to administration of suppression agents. Levels of expression of TRH-DE at the site of administration of suppression agents targeting TRH-DE can be undertaken using inter alia (rt) PCR, real-time PCR and/or northern blotting to quantify transcript levels and western blotting, fluorometric analyses and immunocytochemistry to evaluate proteins levels.

The present invention comprises methods and agents to increase the bioavailability and/or effects of TRH. The invention is not limited to the examples as set forth herein and it should be understood that various modifications might be included that would fall within the scope and spirit of the present invention. The specification together with the examples provided set forth the preferred embodiments of the invention.

The words "comprises/comprising" and the words "having/including" when used herein with reference to the present invention are used to specify the presence of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

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 sub-combination. References

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Claims

1. A suppression agent for increasing the bioavailability of TRH comprising a nucleic acid comprising a supression sequence at least partially complimentary to or at least partially homologous to a portion of TRH-DE RNA.

2. A suppression agent as claimed in claim 1 wherein the TRH-DE RNA comprises a sequence at least partially complementary to at least a portion of one or more of SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15 and SEQ ID NO 16.

3. A suppression agent as claimed in claim 1 or 2 wherein the supression sequence is about 15 bases to about 100 bases in length

4. A suppression agent as claimed in any one of claims 1-3 wherein the supression sequence is about 15 to about 27 bases in length.

5. A suppression agent as claimed in any one of claims 1-4 wherein the nucleic acid is an an siRNA molecule.

6. A suppression agent as claimed in any one of claims 1-4 wherein the nucleic acid is an antisense oligonucleotide.

7. A suppression agent as claimed in any one of claims 1-5 wherein the nucleic acid is a microRNA molecule.

8. A suppression agent as claimed in any of claims 1-7 wherein the supression sequence is at least partially complimentary to or at least partially homologous to one or more of SEQ ID NOs 1-7 or the sequences shown in Table 2.

9. A suppression agent as claimed in any one of claims 1-3 wherein the nucleic acid is a ribozyme.

10. A suppression agent as claimed in any of claims 1 to 7 or claim 9 wherein the supression sequence is at least partially complimentary to or at least partially homologous to SEQ ID NO lO.

11. A composition comprising one or more suppression agents as claimed in any one of claims 1-10.

12. A composition as claimed in claim 11 further comprising a TRH or a TRH like peptide.

13. A composition as claimed in any one of claims 11 or 12 further comprising one or more inhibitors of TRH-DE.

14. A composition as claimed in any one of claims 11-13 further comprising a vector for delivering the suppression agent to cells, tissues or physiological locations.

15. A pharmaceutical composition comprising a suppression agent of any of claims 1 - 10 or a composition as claimed in any one of claims 11-14.

16. A pharmaceutical composition as claimed in claim 15 further comprising a pharmaceutical carrier.

17. Use of a suppression agent of any one of claims 1-10 or a composition of any one of claims 11 - 16 in the preparation of a medicament for the treatment of a disorder of the CNS.

18. A method of treating a disorder of the CNS comprising administering a suppression agent of any of claims 1-10 or a composition as claimed in any one of claims 11-16 to a patient.

19. Use as claimed in claim 17 or a method as climed in claim 18 wherein the disorder of the CNS is selected from the one or more of the group consisting of, spinal injury, memory loss, spinocerebellar degeneration, spinal cord pain, epilepsy, obesity, diabetes, psychiatric disorders, disorders of mood, Attention Deficit Disorder, jet lag, ADHD, AD and CNS related diseases.

20. Use of a suppression agent as claimed in any one of claims 1-10 or a composition as claimed in any one of claims 11-16 in one or more of the inhibition of TRH-DE expression, increasing the bioavailability of TRH, as a research tool to investigate TRH- related cellular processes, as a research tool to investigate TRH-DE-related cellular processes, in the generation of transgenic cells and in the generation of transgenic animals.

21. A suppression agent, composition, use thereof or method of treating a patient substantially as herein described with reference to the description and accompanying figures, tables and sequences.

TOMKINS & CO.