WO2002097092A1 - Neuronal cell-specific promoter - Google Patents

Abstract

A polynucleotide is provided comprising a nucleotide sequence corresponding to the 5' promoter region of a glycine receptor alpha1 subunit gene or a derivative, variant or fragment of said sequence capable of conferring neuron-specific expression of a heterologous nucleotide sequence operably linked thereto.

Description

NEURONAL CELL-SPECIFIC PROMOTER

Field of the invention

The present invention relates to a promoter which confers neuron-specific expression. The promoter may be used to direct expression of therapeutic gene products specifically in neurons, such as motor neurons.

Background to the invention

Traumatic spinal cord damage results in severe and lifelong disability and, in the worst instance, leads to irreversible paralysis below the site of injury. Reversing the permanence of paralysis remains one of the greatest challenges in medical science, as no treatment for the recovery of motor functions after spinal cord injury is presently available. Progress made in spinal cord research over the last decade has started to shed some light into the cellular and molecular mechanisms underlying neuronal survival and axonal regeneration. For instance, there is now good evidence that neurotrophic factors, which are proteins that promote the growth of axons and that contribute to neuronal maturation during the ontogenesis of the central nervous system (CNS), can assist the regeneration of the mature central nervous system. Indeed, some members of the neurotrophin family such as glial-cell-line-derived neurotrophic factor (GDNF) and neurotrophin-3 (NT-3) have been shown to stimulate axon collateral sprouting and prevent the axotomy-induced death of corticospinal neurons. More importantly, it has been recently demonstrated that these neurotrophins can stimulate the recovery of motor functions (Ramer et al., 2000, Nature 403: 312-316). However, systemic administration of GDNF or NT-3 is of limited value since it has been reported that the dose required to generate a therapeutic effect is above the toxicity threshold in humans.

The CNS is a soft structure that is guarded against possible mechanical damage by the skull and the vertebrae as well as by the meninges. Additionally, the

CNS relies on the blood-brain barrier for its defence against the presence of foreign agents that could be present in the bloodstream. These physical and chemical protective mechanisms hardly make the CNS amenable to clinical treatment. However, spinal cord motor neurons innervate the skeletal musculature with their peripherally projecting axons, and this offers an indirect route of administration of therapeutic agents. Gene therapy has taken advantage of this distinctive feature of motor neurons to deliver neurotrophic factors to the spinal cord. Indeed, it is possible to transduce motor neurons with neurotrophic factors by injecting vectors containing the genes that encode for these factors into the peripheral musculature. When injected in a muscle, the vectors carrying neurotrophic factor genes are taken up by the axon terminals that are present at the injection site and the therapeutic agents are subsequently transported, in a retrograde fashion, to the motor neurons from which these axons originate.

The delivery and expression of genetic material can also be achieved with viral systems such as adenoviral vectors and non-viral gene delivery systems such as cationic liposomal DNA preparations. Non-viral systems have a number of advantages over viral systems. For example, they can be produced rapidly and at low cost. Moreover, unlike viral constructs, plasmid constructs are not limited by the size of the transgene they carry. Over the last decade, successful in vivo and in vitro cationic lipid-mediated DNA transfection (i.e., lipofection) of nerve cells has been repeatedly reported.

A number of groups have demonstrated that injection of naked DNA, i.e., without any special delivery system, into the skeletal musculature results in the uptake and sustained expression of the transgene in the muscle cells. Relatively efficient gene transfer in the CNS has also been achieved by direct injections of naked DNA. In the CNS, the transduction obtained with unconjugated DNA has been reported to be stable, although lower than that produced with other gene vehicles such as viral vectors or DNA plasmids complexed with cationic lipid. Gene delivery by means of a non-viral vector has recently attracted considerable interest with the development of an effective therapeutic DNA vaccines approach (Huang et al., 1999, Neuron 24: 639-647; for a review see Gurunathan et al., 2000, Ann. Rev. Immunol. 18: 927-974).

The viral cytomegalovirus (CMV) promoter has been widely used to direct the expression of different transgenes in the CNS. However, the use of vectors containing the CMV promoter also results in the expression of genes in non- neuronal elements such as astrocytes, macrophages, and endothelial cells. Alternatively, other promoters such as the rat neuron-specific enolase (NSE), the human synapsin 1 (SYN), and the rat tubulin αl (Tal) gene promoters have also been used to limit the expression of transgene to neuronal cells (e.g., Twyman and Jones, 1997, J. Mol. Neuroscience 8: 63-73; Kϋgler et al., 2000, Mol. Cell. Neuroscience 17: 78-96).

One of the great challenges of gene therapy is the transfer of therapeutic genes into the precise region of the CNS where the injury resides. This issue is particularly critical for the treatment of paralysis resulting from traumatic spinal cord injury, as the delivery of neurotrophic factors under the control of a non-neuron specific promoter in the peripheral musculature can have unpredictable effects. Although it is clear that an ideal promoter should be cell-specific, no successful attempt has been made previously to design a nucleotide vector that is under the control of a motor neuron specific promoter.

Summary of the invention

We have demonstrated that an approximately 5 kb fragment of a glycine receptor alphal subunit (GlyR alphal) gene promoter confers neuronal cell- specific expression on a heterologous nucleotide sequence to which it is operably linked.

Accordingly, the present invention provides a polynucleotide comprising a nucleotide sequence corresponding to the 5' promoter region of a glycine receptor alphal subunit gene or a derivative, variant or fragment of said sequence capable of conferring neuron-specific expression of a heterologous nucleotide sequence operably linked thereto.

The present invention also provides a polynucleotide comprising a nucleotide sequence as shown in SEQ ID No. 1, or a homologue, derivative, variant or fragment of said sequence capable of conferring neuron-specific expression of a heterologous nucleotide sequence operably linked thereto. Particularly preferred fragments are nucleotides 1 to 5397 and nucleotides 2200 to 5397 of SEQ ID No. 1, or the equivalent regions in other GlyRalphal promoter sequences.

Preferably said neuron-specific expression is specific to motor neurons and/or neurons which express glycine receptors, which include motor neurons and interneurons.

Typically the polynucleotides of the invention are operably linked to a heterologous nucleic acid of interest (NOI) such that the polynucleotide directs expression of the NOI in a neuron, preferably a motor neuron and/or a neuron which expresses a glycine receptor, which includes motor neurons and interneurons.

The NOI may encode a polypeptide of therapeutic use such as a polypeptide which is cytotoxic, a polypeptide capable of converting a precursor prodrug into a cytotoxic compound or a polypeptide selected from polypeptides involved in the regulation of cell division, enzymes involved in cellular metabolic pathways, neurotrophic factors, transcription factors and agents such as antibodies or polypeptides which block the action of inhibitory factors.

The present invention also provides a nucleic acid vector comprising a polynucleotide of the invention. Polynucleotides and nucleic acid vectors of the invention may be combined with a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical composition.

Polynucleotides, nucleic acid vectors and pharmaceutical compositions of the invention may be used to deliver therapeutic genes to mammalian neurons, preferably motor neurons and/or neurons that express a glycine receptor, which include motor neurons and interneurons. More particularly, polynucleotides, nucleic acid vectors and pharmaceutical compositions of the invention may be used to treat patients having conditions characterised by disease of and/or injury to the central nervous system (CNS). However, a number of disorders that may originate in the CNS only present symptoms in the other tissues, including the peripheral nervous system (PNS) and non-neural tissue. Thus patients with other disorders, such as disorders associated with, but necessarily of, the CNS may also be treated where delivery of therapeutic genes to the CNS is of benefit.

Accordingly, the present invention provides a polynucleotide, nucleic acid vector and pharmaceutical composition of the invention for use in a method of treatment of a human or animal.

Also provided is a method of treatment of a human or animal patient suffering from a disease of, or injury to, the central nervous system, or associated with the central nervous system which method comprises administering an effective amount of a polynucleotide, a nucleic acid vector or a pharmaceutical composition of the invention to the patient in need of such treatment.

In a preferred embodiment, the polynucleotide, nucleic acid vector, viral vector or pharmaceutical composition is administered to the human or animal by injection into the tongue muscle of the human or animal. More preferably, the nucleic acid vector, viral vector or pharmaceutical composition is administered by non- viral means. We have taken advantage of the direct innervation of individual muscles by glycine receptor-expressing spinal cord motor neurons to develop a system that uses retrograde axonal transport for delivery of genes specifically to glycine receptor-expressing neurons. We have shown for the first time that nucleic acid constructs free of complexing agents, such as cationic polymers are successfully transported to the CNS, in particular motor neurons. This system can therefore be used to direct the expression of therapeutic gene products specifically to brainstem and spinal cord motor neurons. The use of naked DNA in pharmaceutical compositions is advantageous compared with complexed DNA since it is difficult to obtain complexes of DNA with delivery vehicles such as cationic polymers that have predictable properties, as is required for pharmaceutical compositions. Accordingly, the demonstration herein for the first time that naked DNA can be successfully delivered to neuronal cells by retrograde axonal transport will assist in the preparation of suitable pharmaceutical formulations for the delivery of nucleic acids to neuronal cells.

Accordingly, in a further aspect, the present invention provides a method of delivering a nucleic acid to a neuronal cell by non-viral means which method comprises administering said nucleic acid as a naked nucleic acid substantially free of complexing agents, to muscle tissue such that the nucleic acid is transported to said neuronal cell by retrograde axonal transport. Preferably the nucleic acid comprises a promoter of the invention operably linked to a heterologous sequence encoding a gene product of interest.

We have also developed a system for identifying new sequences that direct expression specifically in neuronal cells and further characterising sequences such as the GlyR alphal promoter disclosed herein.

Thus, in a further aspect, the present invention also provides a method for determining whether a candidate nucleotide sequence is capable of confering neuron-specific expression of a nucleotide sequence of interest operably linked thereto which method comprises: (i) providing a non-viral nucleotide vector comprising said candidate nucleotide sequence operably linked to a nucleic acid of interest; (ii) administering said vector to the brain or brain stem of a non- human animal; and (iii) determining whether said nucleotide sequence of interest is expressed specifically in neurons in the brain or brain stem of said animal.

Preferably said neurons are motor neurons and/or glycine receptor-expressing neurons. Preferably said specificity is in glycine receptor-expressing neurons.

The present invention also provides nucleotide sequences obtained by the above method of the invention, which sequence are capable of confering neuron-specific expression of a nucleic acid of interest operably linked thereto, particularly motor neuron-specific expression and/or expression specific to glycine receptor expressing cells.

Detailed description of the invention

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g. in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry). Standard techniques are used for molecular, genetic and biochemical methods (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2^nd ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (1999) 4^th Ed, John Wiley & Sons, Inc. - and the full version entitled Current Protocols in Molecular Biology, which are incorporated herein by reference) and chemical methods.

A. Polynucleotides

Polynucleotides of the invention may comprise DNA or RNA. They may be single-stranded or double-stranded. They may also be polynucleotides which include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the present invention, it is to be understood that the polynucleotides described herein may be modified by any method available in the art. Such modifications may be carried out, for example, to enhance the in vivo activity or life span of polynucleotides of the invention.

Polynucleotides of the invention are typically in an "isolated" form. This means that are not in their naturally occurring form i.e. polynucleotides of the invention do not include GlyRalphal promoters present in the genome from which they originate together with the coding sequences which they normally direct expression of. Thus, when a polynucleotide of the invention is present in a host cell, it is typically heterologous to that cell.

Promoter sequences

Polynucleotides of the invention comprises a sequence based on the 5' promoter region of a glycine receptor alphal (GlyRalphal) subunit. Such a sequence may correspond directly to the 5' promoter region of a naturally occurring GlyRalphal gene (such as the human GLRAl gene or mouse Glτal gene) or it may be a variant or derivative thereof as described below. GlyRalphal genes have been identified in a number of organisms including humans and mice. Consequently, a GlyRalphal promoter according to the present invention from may be obtained from the 5¹ promoter region of any GlyRalphal gene from any organism whose genome comprises such a gene. Typically the GlyRalphal promoter will be of mammalian origin, such as mouse, rat or primate, more preferably human.

In a preferred embodiment, the GlyRalphal promoter of the present invention comprises the nucleotide sequence shown as SEQ ID NO. 1, or a homologue, derivative, variant or fragment thereof. The terms "variant", or "derivative" in relation to the GlyRalphal promoter nucleotide sequence of the present invention includes any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleotides from or to the sequence provided that the resultant nucleotide sequence is capable of conferring neuron-specific expression of a heterologous nucleotide sequence operably linked thereto, preferably motor neuron and/or glycine receptor expressing neurons. Preferably, such a variant, or derivative directs expression to at least 50% of the levels obtained with the polynucleotide sequence of SEQ ID No. 1, preferably at least 75% or 90%. Nucleotide sequences where large numbers of changes have been made such that the sequence is no longer recognisable to a person skilled in the art a promoter sequence of the invention are not intended to be encompassed by the terms "variant" and "derivative". Accordingly where variants and derivatives are produced by modifying existing sequences, typically less than 10 or 20% of the sequence is modified, preferably less than 5%. For example, it is preferred that fewer than 100, 50 or 25 nucleotides be altered in a sequence having 1000 nucleotides.

However, as discussed below, this limits typically apply only to those regions containing sequences that bind transcriptional components and therefore are responsible for transcriptional regulation. A person skilled in the art will appreciate that regions of the promoter sequence shown in SEQ ID No.l for example are irrelevant to the functioning of the promoter and may be modified as desired. Examples of such regions include the repetitive repeat regions described in Tables 3 and 4.

With respect to homologous sequences preferably there is at least 75%, more preferably at least 85%, more preferably at least 90% homology to the sequences shown in the SEQ ID NO. 1. More preferably there is at least 95%, more preferably at least 98%, homology.

Calculation of % homology may be carried out using, for example, computer software that generates an optimum alignment and then produces a homology score such as the GGG Wisconsin Bestfit package (University of Wisconsin, U.S.A.; Devereux et al., 1984, Nucleic Acids Research 12:387). Examples of other software than can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al., 1999 ibid - Chapter 18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al., 1999 ibid, pages 7-58 to 7-60).

However, the preferred sequence comparison program is the GCG Wisconsin Bestfit program described above using the program defaults. The default scoring matrix has a match value of 10 for each identical nucleotide and -9 for each mismatch. The default gap creation penalty is -50 and the default gap extension penalty is -3 for each nucleotide.

Promoter sequences comprise particular motifs (promoter elements) which act as binding sites for transcription factors that regulate transcription from the promoter. The sequence of these sites is more important than flanking "spacer" regions for the activity of the promoter. Consequently, modifications to the promoter sequence should avoid changes to the sequence of transcription factor binding motifs which would significantly affect the binding of the corresponding transcription factor. However, it is often possible to replace particular promoter elements from the promoter of one gene with the an equivalent element from another gene, which binds the same transcription factor. Such motif "swaps" are within the scope of the present invention.

It may also be desirable to modify GlyRalphal promoters of the present invention to increase or decrease the levels of expression in neurons such as glycine receptor expressing cells. This may, for example be achieved by increasing the number of promoter elements to allow binding of additional transcriptional activator proteins and/or by making substitutions in motifs, such as motifs that bind general transcriptional components of the RNA polymerase π transcription machinery, to provide a stronger consensus sequence. For example, the proximal promoter elements just upstream from the transcriptional initiation site (i.e. up to about 200 to 300 nucleotides 5' of the transcriptional initiation site, which is at nucleotide 5399 of SEQ ID No. 1) may be replaced with other promoter elements that function in a similar manner.

In addition, a consequence of the way in which promoters are built up from various promoter elements is that overall homology comparisons may be less useful than comparisons of particular regions containing sequence elements. It is therefore preferred that homologous sequences have higher homology in these regions whereas intervening regions may have much lower homology. In this regard, Table 4 in the Examples indicates the location in SEQ ED No. 1 of various regions of repetitive DNA. These regions are unlikely to contain promoter elements that confer cell-type specificity. Consequently, when carrying out homology comparisons, these regions are likely to be of low importance. Equally, when modifying GlyRalphal promoter sequences of the present invention, substitutions, deletions and insertions in these regions may not affect to any significant extent the cell-type specificity of the promoter sequences of the invention.

The present invention also encompasses nucleotide sequences that are capable of hybridising selectively to the sequences presented herein. The term "selectively hybridisable" means that the polynucleotide used as a probe is used under conditions where a target polynucleotide of the invention is found to hybridise to the probe at a level significantly above background. The background hybridisation may occur because of other polynucleotides present, for example, in the cDNA or genomic DNA library being screening. In this event, background implies a level of signal generated by interaction between the probe and a nonspecific DNA member of the library which is less than 10 fold, preferably less than 100 fold as intense as the specific interaction observed with the target DNA. The intensity of interaction may be measured, for example, by radiolabelling the probe, e.g. with ³²P.

Hybridisation conditions are based on the melting temperature (Tm) of the nucleic acid binding complex, as taught in Berger and immel (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol 152, Academic Press, San Diego CA), and confer a defined "stringency" as explained below.

Maximum stringency typically occurs at about Tm-5°C (5°C below the Tm of the probe); high stringency at about 5°C to 10°C below Tm; intermediate stringency at about 10°C to 20°C below Tm; and low stringency at about 20°C to 25°C below Tm. As will be understood by those of skill in the art, a maximum stringency hybridisation can be used to identify or detect identical polynucleotide sequences while an intermediate (or low) stringency hybridisation can be used to identify or detect similar or related polynucleotide sequences.

In a preferred aspect, the present invention covers nucleotide sequences that can hybridise to the nucleotide sequence of the present invention under stringent conditions (e.g. 65°C and O.lxSSC {lxSSC = 0.15 M NaCl, 0.015 M Na₃ Citrate pH 7.0}).

In a preferred embodiment, the GlyRalphal promoter sequence of the invention comprises less than 10000 nucleotides preferably fewer than 8000 or 6000 nucleotides. Smaller fragments are also within the scope of the invention, such as fragments having less than 5000, 4000, 3000, 2000 or 1000 nucleotides, provided that such fragments are capable of conferring neuron-specific expression of a heterologous sequence operably linked thereto, preferably motor neuron-specific expression and/or glycine receptor expressing neuron- specific expression. Preferred fragments comprise at least 200, 300, 400, 500 or 700 nucleotides. Highly preferred fragments comprise nucleotides 1 to 5397 (sequence 5' of the transcriptional start site) or nucleotides 2200 to 5397 of SEQ ID No. 1, or the equivalent regions in other GlyRalphal promoter sequences. Other preferred fragments may lack nucleotides 4621 to 5720 of the sequence shown as SEQ ID No. 1, or equivalent regions of homologues, variants and derivatives thereof. Consequently a preferred fragment consists essentially of nucleotides 1 to 4620 or 2200 to 4620 of SEQ ID No. 1, or the equivalent regions in other GlyRalphal promoter sequences.

The minimal region required to confer neuron-specific expression may be determined by progressively deleting regions of, for example, the sequence shown as SEQ ID No. 1 and testing for specificity of expression using the expression assay described below which involves injection of constructs into the brain or brain stem of non-human animals, such as mice.

In addition to the GlyRalphal promoter sequence of the present invention, the polynucleotide of the invention may comprise additional regulatory control sequences. For example, additional levels of transcriptional control may be used to ensure that expression directed by the promoter sequence of the invention is confined to certain cells under certain conditions. Thus, for example, additional enhancers may be operably linked to the GlyRalphal promoter sequence of the invention, either downstream, upstream or both.

The additional regulatory sequence may be a sequence found in eukaryotic genes. For example, it may be a sequence derived from the genome of a cell in which expression of directed by the promoter of the invention is to occur. It one embodiment the additional regulatory sequence is not a sequence that is naturally found operably linked to the promoter sequence of the present invention.

In most instances, these additional regulatory sequences, such as enhancers may be isolated as convenient restriction digestion fragments suitable for cloning in a selected vector. Alternatively, regulatory sequences may be isolated using the polymerase chain reaction. Cloning of the amplified fragments may be facilitated by incorporating restriction sites at the 5' end of the primers. Regulatory sequences may also be synthesised using, for example, solid-phase technology. It may also be desirable to include regulatory elements that are inducible, for example such that expression can be regulated by administration of exogenous substances. In this way, levels of expression of directed by the promoter can be regulated during the life-time of the cell. Inducible means that the levels of expression obtained using the promoter can be regulated. For example, in a polynucleotide of the invention may comprises regulatory sequences responsive to the tet repressor VPl6 transcriptional activator fusion protein. Thus in this example, expression directed by the promoter of the invention would depend on the presence or absence of tetracycline.

An advantageous feature of the promoter sequences of the present invention is that they are neuron cell-specific. In particular, they are specific for cells that express strychnine sensitive glycine receptors comprising an alpha subunit. The term "cell specific" means a regulatory control sequence which is not necessarily restricted in activity to a single cell type but which nevertheless shows selectivity in that it is active in one group of cells and less active or silent in another group. However, it may be preferred that promoters of the invention show strict cell-specificity in that they are only active at detectable levels in neuronal cells, such as cells that express glycine receptors.

Preferred neuronal cells in which promoters of the present invention show specificity of expression are cells that express strychnine-sensitive glycine receptors comprising alpha sub units. Glycine receptors (GlyRs) are almost exclusively found on the cell surface of motor neurones located in the brainstem as well as throughout the full length of the spinal cord.

Nucleic acids of Interest (NOI

Polynucleotides comprising a promoter of the invention are typically operably linked to an NOI, usually a heterologous gene. The term "heterologous gene" encompasses any gene. The heterologous gene may be any allelic variant of a wild-type gene, or it may be a mutant gene. The term "gene" is intended to cover nucleic acid sequences which are capable of being at least transcribed. Thus, sequences encoding mRNA, tRNA and rRNA are included within this definition. The sequences may be in the sense or antisense orientation with respect to the promoter. Antisense constructs can be used to inhibit the expression of a gene in a cell according to well-known techniques. Nucleic acids may be, for example, ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or analogues thereof. Sequences encoding mRNA will optionally include some or all of 5' and/or 3' transcribed but untranslated flanking sequences naturally, or otherwise, associated with the translated coding sequence. It may optionally further include the associated transcriptional control sequences normally associated with the transcribed sequences, for example transcriptional stop signals, polyadenylation sites and downstream enhancer elements. Nucleic acids may comprise cDNA or genomic DNA (which may contain introns). However, it is generally preferred to use cDNA because it is expressed more efficiently since intron removal is not required.

The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequence.

In accordance with the present invention, suitable NOI sequences include those that are of therapeutic and/or diagnostic application such as, but are not limited to: sequences encoding cytokines, chemokines, hormones, antibodies, engineered immunoglobulin-like molecules, a single chain antibody, fusion proteins, enzymes, immune co-stimulatory molecules, immunomodulatory molecules, anti-sense RNA, a transdominant negative mutant of a target protein, a toxin, a conditional toxin, an antigen, a tumour suppressor protein and growth factors, membrane proteins, anti-viral proteins and ribozymes, and derivatives therof (such as with an associated reporter group). NOI(s) may be used which encode polypeptides, antibodies, antisense transcripts or ribozymes which interfere with expression of cellular genes, such as inhibitory proteins that inhibit the action of neurotrophins.

Suitable NOIs for use in the present invention may also include NOIs that may be used in the treatment or prophylaxis of cancer include NOIs encoding proteins which: destroy the target cell (for example a ribosomal toxin), act as: tumour suppressors (such as wild-type p53); activators of anti-tumour immune mechanisms (such as cytokines, co-stimulatory molecules and immunoglobulins); or which provide enhanced drug sensitivity (such as pro- drug activation enzymes); indirectly stimulate destruction of target cell by natural effector cells (for example, strong antigen to stimulate the immune system or convert a precursor substance to a toxic substance which destroys the target cell (for example a prodrug activating enzyme). Encoded proteins could also destroy bystander tumour cells (for example with secreted antitumour antibody-ribosomal toxin fusion protein), indirectly stimulated destruction of bystander tumour cells (for example cytokines to stimulate the immune system or procoagulant proteins causing local vascular occlusion) or convert a precursor substance to a toxic substance which destroys bystander tumour cells (e.g. an enzyme which activates a prodrug to a diffusible drug).

Instead of, or as well as, being selectively expressed in target tissues, the NOI or NOIs may encode a pro-drug activation enzyme or enzymes which have no significant effect or no deleterious effect until the individual is treated with one or more pro-drugs upon which the enzyme or enzymes act. In the presence of the active NOI, treatment of an individual with the appropriate pro-drug leads to enhanced reduction in tumour growth or survival.

In each case, a suitable pro-drug is used in the treatment of the patient in combination with the appropriate pro-drug activating enzyme. An appropriate pro-drug is administered in conjunction with the NOI. Examples of pro-drugs include: etoposide phosphate (with alkaline phosphatase); 5-fluorocytosine (with cytosine deaminase); doxorubicin-N-p-hydroxyphenoxyacetamide (with penicillin- V-amidase); para-N-bis(2-chloroethyl) aminobenzoyl glutamate (with carboxypeptidase G2); cephalosporin nitrogen mustard carbamates (with β-lactamase); SR4233 (with P450 Reducase); ganciclovir (with HSV thymidine kinase); mustard pro-drugs with nitroreductase and cyclophosphamide (with P450).

Examples of suitable pro-drug activation enzymes for use in the invention include a thymidine phosphorylase which activates the 5-fluoro-uracil pro- drugs capcetabine and furtulon; thymidine kinase from herpes simplex virus which activates ganciclovir; a cytochrome P450 which activates a pro-drug such as cyclophosphamide to a DNA damaging agent; and cytosine deaminase which activates 5-fluorocytosine. Preferably, an enzyme of human origin is used.

The expression products encoded by the NOIs may be proteins which are secreted from the cell. Alternatively the NOI expression products are not secreted and are active within the cell.

Particularly preferred NOIs encode neurotrophic factors, such as nerve growth factor (NGF), β-NGF, ciliary neurotrophic factor (CNTF) brain-derived neurotrophic factor (BNTF), glial cell line derived neurotrophic factor (GDNF) and neurotrophins such as NT-3, NT-4, NT-5. For example, it has been shown that delivery of the NT-3 gene has led to an improvement in motor functions in a mouse model (Haase et al., 1997, Nat. Med 3: 380-381).

Preferred NOIs may also encode transcription factor Brn-3a (or an N-terminal fragment thereof) which has been shown to protect neuronal cells from apoptosis (WO99/05202). Brn-3a activates specifically expression of the Bcl-2 gene in neuronal cells: this activation is mediated via a Brn-3a response element in the 5' regulatory region of the Bcl-2 gene.

The NOI may encode antibodies such as IN-1 that neutralise the action of Nogo-A, one of the major inhibitory proteins expression in CNS myelin (Chen et al., 2000, Nature 403: 434-439), or antibodies or other proteins or reagents that inhibit classes of molecules such as the semaphorins, ephrins, etc which are know to have inhibitory or repulse effects on axonal regrowth. The therapeutic use of antibodies to Nogo-A is described in Merkler et al, 2001, J. Neuroscience 21: 3665-3673.

The use of combinations of the above NOIs is also envisaged.

NOIs may also include marker genes (for example encoding beta-galactosidase or green fluorescent protein) or genes whose products regulate the expression of other genes (for example, transcriptional regulatory factors including the tet repressor/VPl6 transcriptional activator fusion protein described above). In addition, NOIs may comprise sequences coding fusion protein partners in frame with a sequence encoding a protein of interest. Examples of fusion protein partners include the DNA binding or transcriptional activation domain of GAL4, a 6xHis tag and beta-galactosidase. It may also be desirable to add targeting sequences to target proteins encoding by NOIs to particular cell compartments or to secretory pathways. Such targeting sequences have been extensively characterised in the art.

Nucleic Acid Vectors

Polynucleotides of the invention can be incorporated into a recombinant vector, typically a replicable vector. The vector may be used to replicate the nucleic acid in a compatible host cell. Thus in a further embodiment, the invention provides a method of making polynucleotides of the invention by introducing a polynucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector. The vector may be recovered from the host cell. Suitable host cells include bacteria such as E. coli, yeast, mammalian cell lines and other eukaryotic cell lines, for example insect Sf9 cells.

A vector comprising a polynucleotide of the invention which is operably linked to an NOI can be considered to be an expression vector since under suitable conditions, the NOI will be expressed under the control of the promoter construct of the present invention. However, it is not necessary for a vector of the invention to comprise an NOI. Nonetheless it is possible to introduce an NOI into the vector at a later stage. Thus a vector of the invention which lacks an NOI can be considered to be a cloning vector. Preferably, a cloning vector of the invention comprises a multiple cloning site downstream of the GlyRalphal promoter sequences to enable an NOI to be cloned into the vector when required whereby it is then operably linked to the GlyRalphal promoter sequences.

The vectors may be for example, plasmids, chromosomes, artificial chromosomes or virus vectors. The vectors may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid or a neomycin resistance gene for a mammalian vector. Vectors may be used, for example, to transfect, transform or transduce a host cell either in vitro or in vivo.

D. Delivery of nucleic acid vectors to neuronal cells

Nucleic acid vectors of the present invention may be delivered to neuronal cells, such as motor neurons and/or glycine receptor-expressing neurons by viral or non- viral means. However, it is preferred to use non- viral means.

Non-viral delivery systems include but are not limited to DNA transfection methods. Here, transfection includes a process using a non- viral vector to deliver a gene to a target mammalian cell. Typical transfection methods include electroporation, DNA biolistics, lipid-mediated transfection, compacted DNA-mediated transfection, liposomes, immunoliposomes, lipofectin, cationic agent-mediated, cationic facial amphiphiles (CFAs), multivalent cations such as spermine, cationic lipids or porylysine, 1, 2,-bis (oleoyloxy)-3- (trimethylammonio) propane (DOTAP)-cholesterol complexes and combinations thereof. Alternatively, polynucleotides/nucleic acid vectors of the invention may be naked polynucleotide constructs in the sense that they may be free from any delivery vehicle, which would act to facilitate entry into the cell (e.g. viral sequences) and/or to promote transfection (e.g., liposomes, polybrene, divalent cations).

It is preferred to use such naked polynucleotide constructs to avoid complications and side effects that may arise from the use of delivery vehicles. Further, the use of naked DNA in pharmaceutical compositions is advantageous compared with complexed DNA since it. is difficult to obtain complexes of DNA with delivery vehicles such as cationic polymers that have predictable properties, as is required for pharmaceutical compositions.

Preferably, the sequences used in the method of the invention do not integrate into the genome of the host cell, but rather remain in the cell as episomal elements.

Viral delivery systems include but are not limited to an adenovirus vector, an adeno-associated viral (AAV) vector, an alphavirus vector, a herpes viral vector, a retroviral vector, such as a lentiviral vector and combination vectors such as an adenolenti viral vector. In the case of viral vectors, gene delivery is typically mediated by viral infection of a target cell.

Generally, target cells will be present in a living multicellular organism. Administration may be by direct introduction into the site to be treated, for example by injection into the brain, brain stem or spinal cord. Alternatively, administration may be by indirect means such as by injection into the tongue or other muscle. The polynucleotides may then reach the central nervous system by axonal retrograde transport to neuronal cell bodies in the brain stem.

E. Therapeutic uses and administration The polynucleotides, vectors and compositions of the present invention may be used to treat diseases of the nervous system which affect neurons such as motor neurons. For example, the polynucleotide, nucleic acid vectors of the invention may be used to deliver therapeutic genes to a human or animal in need of treatment.

Diseases which may be treated, prevented or alleviated include diseases of the central nervous system such as neurodegenerative diseases and damage to nervous tissue as a result of injury/trauma (including strokes and spinal cord injuries).

However, a number of disorders that may originate in the CNS only present symptoms in the other tissues, including the peripheral nervous system (PNS) and non-neural tissue. Thus patients with other disorders, such as disorders associated with, but necessarily of, the CNS may also be treated where delivery of therapeutic genes to the CNS is of benefit.

Specific diseases or conditions that may be treated include inflammatory components of stokes, post-polio syndrome, acute neuropathy, subacute neuropathy, chronic neuropathy, pseudo-tumour cerebri, Huntington's disease, amyotrophic lateral sclerosis, inflammatory components of CNS compression or CNS trauma or infections of the CNS, conditions or disorders of the central and peripheral nervous systems. In particular, neurodegenerative diseases include motor neuron disease, several inherited diseases, such as familial dysautonomia and infantile spinal muscular atrophy, hyper-ekplexia, and late onset neurodegenerative diseases such as Parkinson's and Alzheimer's diseases.

The polynucleotides, vectors and compositions of the present invention which express therapeutic genes may be used to stimulate the growth of axons, neuronal maturation and/or neuronal regeneration.

The polynucleotides of the invention may be administered directly to a patient in need of therapy as a naked nucleic acid construct. Uptake of naked nucleic acid constructs by mammalian cells is enhanced by several known transfection techniques for example those including the use of transfection agents. Example of these agents include cationic agents (for example calcium phosphate and DEAE-dextran) and lipofectants (for example lipofectam™ and transfectam™ ). Thus nucleic acid constructs may be mixed with the transfection agent to produce a composition. However, it is preferred that compositions of the invention lack nucleic acid complexing agents such as cationic lipids.

Preferably the naked nucleic acid construct or vector is combined with a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical composition. Suitable carriers and diluents include isotonic saline solutions, for example phosphate-buffered saline. The composition may be formulated for parenteral, intramuscular, intravenous, subcutaneous, intraocular or transdermal administration. The pharmaceutical composition may be for human or animal usage.

The pharmaceutical composition is administered in such a way that the polynucleotide of the invention can be incorporated into cells at an appropriate area. For example, when the target of gene therapy is the central or peripheral nervous system and the polynucleotide of the invention is to be delivered by a herpes simplex virus vector, the composition could be administered in an area where synaptic terminals are located so that the virus can be taken up into the terminals and transported in a retrograde manner up the axon into the axonal cell bodies via retrograde axonal transport. The pharmaceutical composition is typically administered to the brain by stereotaxic inoculation. When the pharmaceutical composition is administered to the eye, sub-retinal injection is typically the technique used.

In a preferred embodiment, polynucleotides are administered non-virally via retrograde axonal transport i.e. intramuscular injection of non-viral DNA so that the DNA is taken up and transported to motor neurons by retrograde axonal transport. One technique involves injection into the tongue muscle as described in Wang et al, 2001, Mol. Ther 3: 658-664. However, Wang et al. use DNA complexed/conjugated to cationic polymers. By contrast, we have shown that naked DNA can be injected into the tongue muscle and transported to motor neurons by retrograde axonal transport, without the requirement for complexing agents that may have undesirable physiological side-effects. It is therefore preferred to administer nucleic acids as naked DNA, typically formulated as a composition with a pharmacetically acceptable carrier or diluent.

When the polynucleotide of the invention is delivered to cells by a viral vector, the amount of virus administered is in the range of from 10³ to 10⁹ pfu, preferably from 10⁵ to 10⁷ pfu. When injected, typically 1-10 μl of virus in a pharmaceutically acceptable suitable carrier or diluent is administered. When the polynucleotide/vector is administered as a naked nucleic acid, the amount of nucleic acid administered is typically in the range of from 1 μg to 10 mg, preferably from 100 μg to 1 mg.

Where the polypeptide of the invention is under the control of an inducible promoter, it may only be necessary to induce gene expression for the duration of the treatment. Once the condition has been treated, the inducer is removed and expression of the NOI ceases. This will clearly have clinical advantages. Such a system may, for example, involve administering the antibiotic tetracycline, to activate gene expression via its effect on the tet repressor VP 16 fusion protein.

The use of neuron-specific promoters of the invention will be of assistance in the treatment of diseases of the nervous system. For example, several neurological disorders are due to aberrant expression of particular gene products in only a small subset of cells. It will be advantageous to be able express therapeutic genes in only the relevant affected cell types, especially where such genes are toxic when expressed in other cell types. Examples of neuronal sub-types which may be targeted specifically include motor neurons.

The routes of administration and dosages described are intended only as a guide since a skilled practitioner will be able to determine readily the optimum route of administration and dosage for any particular patient and condition. The efficacy of any given NOI in treating conditions relating to the nervous system may typically be tested using an animal model. For example, it has been shown that delivery of the NT-3 gene has led to an improvement in motor functions in a mouse model (Haase et al., 1997, Nat. Med 3: 380-381). Mouse models of neuronal function are considered in the art to be predictive in assessing the applicability of these treatments to humans.

F. Assays for neuron-cell specific expression

The present invention also provides methods for assaying promoter constructs such as fragments, derivatives and variants of GlyRalpal promoters for activity as neuron-cell specific regulatory control sequences.

These methods comprise providing suitable nucleic acid constructs which comprise a candidate nucleotide sequence whose activity it is desired to test operably linked to a heterologous nucleic acid sequence of interest. The heterologous NOI is selected such that its expression in neuronal cells is detectable. It is especially preferred to use NOIs whose expression can be detected histologically so that the particular cells in which it is expressed can be readily seen in sections of tissue of the CNS. Examples of suitable NOIs include beta-galactosidase and green fluorescent protein or variants thereof (e.g. blue fluorescent protein).

The nucleic acid constructs are administered directly to the brain or brain stem of a non-human animal such as a mouse or a rat using suitable means such as direct injection, for example direct injection into the amygdala (see the Examples).

The animal is then typically allowed to recover and is kept for a period of time, such as 4 to 6 days, to allow retrograde transport of the polynucleotide constructs and expression of the gene product. Animals are then sacrificed and brain and/or brain stem tissue removed for subsequent analysis to determine whether expression of the NOI is specific to neuronal cells and in particular subsets of neuronal cells such as motor neurons and/or glycine receptor expressing cells. This may be achieved, for example, by taking thin sections of brain tissue and visualising products of expression of the NOI by immunohistochemistry and microscopy. Typically, expression specific to glycine receptor-expressing neurons is indicated when expression is seen in the glycine receptor-expressing parabrachial nucleus but not the glycine receptor-lacking insular cortex.

Promoter sequences identified by the above method may be used in polynucleotides constructs of the present invention.

It should be appreciated that features from various sections, aspects and embodiments of the invention as described above are generally equally applicable to other sections, aspects and embodiments mutatis mutandis.

The invention will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention. The Examples refer to the Figures. In the Figures:

Figure 1 EGFP-positive neurons in the hypoglossal nucleus after an injection, in the tongue, of the fusion construct under the control of the 5kb hGlyRαl promoter.

Figure 2 Predicted results from an injection, in the central nucleus of the amygdala (Ce), of the IRES construct under the control of the NSE (upper part) and of the fusion construct under the 5kb hGlyRαl promoters (lower part). The NSE promoter was hypothesised to give rise to EGFP-positive neurons in both the insular cortex (IC) and the parabrachial nucleus (PB). The 5kb hGlyRαl promoter was hypothesised to give rise to EGFP-positive neurons in the PB only.

Figure 3 EGFP-positive neurons in both the insular cortex (IC) (left) and the parabrachial nucleus (PB) (right) after an injection, in the central nucleus of the amygdala (Ce) of the IRES construct under the control of the NSE promoter.

Figure 4 Absence of EGFP-positive neurons in both the insular cortex (IC) (left), but presence of EGFP-positive neurons in the parabrachial nucleus (PB) (right) after an injection, in the central nucleus of the amygdala (Ce) of the fusion construct under the control of the the

5kb hGlyRαl promoter.

EXAMPLES

Construct design

Two types of constructs were generated to evaluate the function and specificity of the glycine receptor 5' promoter. The first type of construct used the pIRES- EGFP vector (Clontech Laboratories Inc, CA, USA) that allows the bicistronic expression of the human glycine receptor alphal subunit (hGlyRalphal) transgene and the enhanced green fluorescent protein (EGFP) reporter gene. With this type of construct the EGFP remains in the cytoplasm of the transfected cells whilst the hGlyRalphal is localised to the cell membrane. The second type of construct, which is based on the pcDNA3.1 (-) plasmid (Invitrogen Corp., NL) expresses the EGFP and the hGlyRalphal as a fusion protein. The fusion protein is expected to behave in the same manner as the native membrane bound receptor. The hGlyRalphal cDNA was subcloned from pCishGlyRalphal (in our laboratory). The 1745 bp insert contains the entire open reading frame, 287 bp of 5' UTR and 115 bp 3' UTR. IRES constructs

In the pIRES-EGFP vector, the Xhol site at 2909bp was deleted and replaced with a Not! site, by insertion of a double stranded linker (GlyRTgXho2). The Nruϊ to EcoRV fragment containing the CMV promoter was excised and the blunt ends ligated (pIX-CMV). A pIRES-EGFP based vector without the Xhol deletion, Notl replacement, and without the CMV promoter removed was also used (pIRES-EGFP-CMV).

The rat neuron-specific enolase promoter was used as a control and l.δkb of promoter was excised from pNSElacZ (gifted by Ora Bernard) with EcoRl and Hindm restriction digest. The 1745 bp of hGlyRalphal coding sequence was amplified with the addition of an EcoRl site and a HinάTH. site, or a Kpήl and a Λfofl site (Table 1).

Human GlyRalphal Gene Promoter

The structure and sequence of the hGlyRalphal promoter is shown in SEQ ID No. 1. The coding region and 5' UTR of hGlyRalphal is shown in SEQ ID No. 2. A region 1097 bases of 5' sequence upstream from the translational start site defined by the sequence ATG, excluding the 5' UTR already contained in pCis hGlyRalphal, was amplified using primers 5' GlyRP2FEco and 3' GlyRPlRevHind with the addition of restriction sites for cloning (Table 1). Human DNA, extracted from blood, was used as a template in the PCR. The hGlyRalphal cDNA was amplified from pCis hGlyRalphal with 5' GlyRPδHind and 3' GlyRP6Eco primers containing restriction sites for cloning. The resulting PCR fragment was digested with EcoRl and Hincttπ and ligated either with the NSE EcoRl-HindUI fragment or the 793 bp GlyR promoter fragment. Ligation products were separated on a 0.8% lx TAE agarose gel. Products of the correct size were excised, purified and cloned into the EcoRl site in PIX-CMV.

The human glycine receptor alphal (hGlyRalphal) subunit gene is located on chromosome 5. From cosmid 77H1, donated to us by Rita Shiang, we determined the DNA sequence of 4620 bp upstream of the published hGlyRalphal promoter sequence. Sequence was confirmed on the reverse strand. In total, 5723 bp upstream of the translational start site was sequenced. This sequence was subjected to a BLAST search to look for expressed sequence tags (ESTs) from other genes. None were found, indicating that the sequence contained only promoter sequences.

A restriction map was constructed and a 3205 bp 5' promoter sequence was PCR amplified with the addition of a Notl site at the 5' end and a .Kp.nl site at the 3' end. The hGlyRalphal cDNA was amplified from pCis hGlyRalphal with primers containing Noil and Kpnl restriction sites for cloning. The resulting PCR fragments were digested with Notl and Kpnl and ligated together. Ligation products were separated on a 0.8% lx TAE agarose gel. Products of the correct size were excised, purified and cloned into pIRES-EGFP-CMV).

Table 1

Primer name Size Sequence 5 '-3'

GlyRTgXho2 14 TCGATGCGGCCGCA (SEQ ID No. 3)

GlyRP2FEco 25 GGGAATTCCGCCAGATCTCGTCCAG (SEQ ID No. 4)

GlyRPlRevHind 30 CCAGCGTGTCAAGCTTCTGCCTGCGGCGCT (SEQ ID No. 5)

GlyRPδNotF 28 GTTTTGGCGGCCGCTATATCCCCAGTGC (SEQ ID No. 6)

GlyRP7NotF 28 GACATGGCGGCCGCCAGCACAGTGTCAG (SEQ ID No. 7)

GlyRPΘ pnF 26 CAGACACGCTGGTACCTAACAAACAG (SEQ ID No. 8)

GlyRPlOKpnR 26 CTGTTTGTTAGGTACCAGCGTGTCTG (SEQ ID No. 9)

GlyRPllNotR 24 GCTTGGGCGGCCGCTCGACTCTAG (SEQ ID No. 10) GlyRPδHindπi 21 ATCAAGCTTGACACGCTGGAG (SEQ ID No. 11)

GlyRP6EcoRI 21 TAGAATTCGCTGCAGGTCGAC (SEQ ID No. 12)

Fusion constructs

Site-directed mutagenesis using primers SacII F and Sacll R were used to introduce a restriction site into hGlyRalphal (in pCis) at nt 381, to enable insertion of the EGFP gene (Table 2). The hGlyRalphal subunit gene was then amplified with primers GlyRPβEcoRI and GlyRP7EcoRI. The PCR product was digested with EcoRl and cloned into the EcoRl site of pcDNA3.1 (-). The ΕGFP gene was amplified by PCR from pIRΕS-ΕGFP with primers ΕGFPSacIIF and ΕGFPSacIIR, and the 717 bp product digested with SacU and subcloned into hGlyRalphal. The CMV promoter was subsequently removed by digesting with Mlul and Xbal and treating the overhangs with Mung bean nuclease. Fragments of the hGlyRalphal promoter differing in size were then cloned into the Notl site in the MCS. Three fragments of the hGlyRalphal promoter were amplified by PCR from the cosmid provided by Rita Shiang. The primers used were; GlyRp2NotF and GlyRPlrNot for the 786 bp product, GlyRPδNotF and GlyRPlrNot for the 3191 kb product, GlyRP7NotF and GlyRPlrNot for the 5412 bp product (Table 1, Table 2). The PCR products were separated on a 0.8% lx TAΕ agarose gel. Products of the correct size were excised, purified and cloned into the modified pcDNA3.1 (-) containing hGlyRalphal fused with ΕGFP.

Table 2

Primer name Size Sequence 5'-3'

GlyRPlrNot 30 CCAGCGTGTGCGGCCGCTCCCTGCGGCGCT (SΕQ ID No. 13)

GlyRP2Fnot 25 GGGGATGCGGCCGCATCTCGTCCAG (SΕQ ID No. 14) GlyRPl pn 29 CCAGCGTGTCTGTTGGTACCGTGCGGCGC (SEQ ID No. 15)

EGFPSacIIR (#6) 26 AGGCCGTGGTGTACAGCTCGTCCATG (SEQ ID No. 16)

EGFPSacIIF (#5) 24 AGGCCGGGGTGAGCAAGGGCGAGG (SEQ ID No. 17)

SacII F 28 CTGCTCGCTCCGCGGCCAAGCCTATGTC (SEQ ID No. lδ)

SacIIR 2δ GACATAGGCTTGGCCGCGGAGCGAGCAG (SEQ ID No. 19)

GlyRP6EcoRI 21 TAGAATTCGCTGCAGGTCGAC (SEQ ID No. 20)

GlyRP7EcoRI 19 ATCGAATTCGACACGCTGG (SEQ ID No. 21)

Example 1 - Retrograde transport and expression of the GlyR gene constructs in the CNS

We initially selected the tongue musculature as the injection site based on the anatomical evidence that neurons in the brainstem hypoglossal nucleus, which is the 12^ cranial motor nucleus, innervate the tongue via the 12*¹¹ cranial nerve. In addition, the muscles of the tongue are easily accessible, so tongue injections provide a simple and relatively non invasive way to evaluate whether injections of our different gene constructs in the peripheral musculature can result in the transport and expression of the reporter gene in the CNS.

Methods

Injections of DNA. C57BL/6 male mice, weighing 17-25 g at the time of surgery, were used in this experiment. Surgery was performed under aseptic conditions. The mice were deeply anaesthetised with a mixture of ketamine (100 mg/ml) and xylazine (20 mg/ml) (80 mg/kg ip) and placed on a heating pad. The animal's tongue was gently pulled out with a pair of forceps and injected with δ μl of DNA (approximately 5 μg in distilled water) through a 0.5 cc insulin syringe (Becton Dickenson, Singapore) at a single point in the midline of the tongue.

The mice were monitored until fully recovered from anaesthesia and then returned to the animal room where they were kept for a minimum of 6 days to allow for the retrograde transport, along the 12^th cranial nerve, and the expression of the reporter gene in the hypoglossal nucleus.

Histology. One week after the surgery, the mice were anaesthetised with, a lethal dose of sodium pentobarbital and perfused through the heart with 0.1 M phosphate buffer saline followed by a solution of paraformaldehyde (4% in 0.1 M phosphate buffer saline). The brainstems were dissected out, post-fixed, embedded in paraffin, and cut into 10 μm-thick coronal sections. The sections were floated in a 42° C water bath and then mounted on microscope slides. The slides were heated at 60° C and subsequently immersed in a histological clearing agent (for 15 min, followed by quick rinses in graded ethanols).

Immunohistochemistry The tissue was rehydrated in 0.1 phosphate buffer saline (PBS), placed in boiling citrate buffer (0.1 M citric acid and 0.1 M trisodium citrate, pH 6.0) for 3 min to retrieve the antigen, washed again in 0.1 PBS, and incubated in a solution of bovine serum albumin and normal goat serum (1% and 10%, respectively in 0.1 PBS) for 20 min. The tissue was then incubated with a rabbit polyclonal antibody raised against the marker protein EGFP (anti-GFP 290, Abeam, Cambridge, UK) (dilution, 1:100 in 0.5% bovine serum albumin and 2% normal goat serum in 0.1 M PBS) for 30 min. After incubation in the primary antibody, the tissue was washed with 0.1 PBS and incubated in the dark for 30 min in Alexa Fluor 4δδ goat anti-rabbit secondary antiserum (Molecular Probe, Eugene, OR, USA) (dilution, 1:100 in 0.5% bovine serum albumin and 2% normal goat serum in 0.1 M PBS). The tissue was subsequently washed in PBS, coverslipped with fluorescent mounting medium (DAKO, CA, USA), and kept in light-tight boxes. Microscopic analysis. The brain tissue was analysed to visualise the EGFP- labelled neurons with a laser scanner confocal microscopr (Leica TCS SP) equipped with a krypton/argon laser. The images were acquired with exitation at 4δδnm and they were exported into Photoshop for presentation.

Results

Figure 1 shows some EGFP-positive neurons in the hypoglossal nucleus resulting from the injection of the EGFP-GlyR fusion construct in the tongue musculature of a C57BL/6 mice. As seen in this figure, the protein marker EGFP is abundant in these neurons, confirming that the injection of the EGFP- GlyR construct in the tongue musculature has resulted in the retrograde transport, along the twelfth cranial nerve, and the expression of the transgene in hypoglossal neurons (see Figure 1). The results of this experiment have therefore established that, when injected in the peripheral musculature, the non- viral delivery system can transfer genes successfully in the CNS and that these constructs can express an NOI in the target neurons.

Both circular plasmid and linearised plasmid were injected, but there was no noticeable difference in EGFP expression.

Example 2 - The specificity of expression of the GlyR gene promoter

To assess the specificity of expression of the GlyR gene promoter, its pattern of expression was compared to that of the rat neuron-specific enolase (NSE) promoter that is known to direct neuron-specific expression of the transgene. GlyRalphal constructs under the control of these two promoters were injected in the central nucleus of the amygdala (Ce) in C57BL/6 mice. The Ce was selected as the brain structure of choice because of its distinctive connectivity with the rest of the CNS. Figure 2 is a diagrammatic representation of the connectivity of the Ce. The Ce receives a projection from both the parabrachial nucleus (PB), a brainstem area in which GlyR are highly expressed, and from the insular cortex (IC) where neurons do not express GlyR. It was hypothesised that an injection of the gene construct that is under the control of the NSE promoter in the Ce would result in the retrograde transport and expression of the transgene in both the IC and the PB. On the other hand, it was predicted that the injection of the gene construct that is under the control of the GlyR gene promoter would be likewise transported to both the IC and the PB, but would only be expressed in the PB (see Figure 2).

Methods

Injections of DNA C57BL/6 male mice, weighing 17-25 g at the time of surgery, were used in this experiment. Surgery was performed under aseptic conditions. The mice were deeply anaesthetised with a mixture of ketamine (100 mg/ml) and xylazine (20 mg/ml) (80 mg/kg ip) and placed on a heating pad, in a stereotaxic apparatus. The head was positioned so that lambda and bregma points were horizontally aligned. An incision was made with a scalpel on the mouse's skin to expose the skull and a small hole was drilled in the skull over the region of interest. The central nucleus of the amygdala was infused over 2 min with 2 μl of DNA (approximately 5 μg in distilled water) through a 33-gauge cannula connected to a lcc syringe (Becton Dickenson, Singapore) driven by an infusion pump (KdScientific, New Hope, PA, USA). The stereotaxic coordinates used for the injections were AP, -1.1; ML, 2.4; DV, -4.0. After the completion of the injections, the cannula was left in place inside the mouse's brain for an additional 5 min to minimise diffusion, after which the wound was cleaned and the skin incision sutured. Topical analgesic ointment (lignocaine and prilocaine, 25 mg/g ) was applied to the wound (Emla, Astra Pharmaceutical, Australia) and the mice were removed from the stereotaxic apparatus and monitored until fully recovered from anaesthesia. The operated mice were then returned to the animal room where they were kept for a minimum of 6 days to allow for the retrograde transport and the expression of the reporter gene.

Histology After the surgery, the mice were anaesthetised with a lethal dose of sodium pentobarbital and perfused through the heart with 0.1 M phosphate buffer saline followed by a solution of paraformaldehyde (4% in 0.1 M phosphate buffer saline). The brains were dissected out, post-fixed, embedded in paraffin, and cut into 10 μm-thick coronal sections. The sections were floated in a 42°C water bath and then mounted on microscope slides. The slides were heated at 60° C and subsequently immersed in a histological clearing agent (for 15 min, followed by quick rinses in graded ethanols. For every injection, tissue sections at the level of the amygdala were stained with Thionin (Sigma, St Louis, MO, USA) and coverslip with DPX mounting medium (BDH Laboratory Supplies, England) to assess the location of the injections.

Immunohistochemistry The tissue was rehydrated in 0.1 phosphate buffer saline (PBS), placed in boiling citrate buffer (0.1 M citric acid and 0.1 M trisodium citrate, pH 6.0) for 3 min to retrieve the antigen, washed again in 0.1 M PBS, and incubated in a solution of bovine serum albumin and normal goat serum (1% and 10%, respectively in 0.1 M PBS) for 20 min. The tissue was then incubated with a rabbit polyclonal antibody raised against the marker protein EGFP (anti-GFP 290, Abeam, Cambridge, UK) (dilution, 1:100 in 0.5% bovine serum albumin and 2% normal goat serum in 0.1 M PBS) for 30 min. After incubation in the primary antibody, the tissue was washed with 0.1 PBS and incubated in the dark for 30 min in Alexa Fluor 488 goat anti-rabbit secondary antiserum (Molecular Probe, Eugene, OR, USA) (dilution, 1:100 in 0.5% bovine serum albumin and 2% normal goat serum in 0.1 M PBS). The tissue was subsequently washed in PBS, coverslipped with fluorescent mounting medium (DAKO, CA, USA), and kept in light-tight boxes.

Microscopic analysis The brain tissue was analysed to visualise the EGFP- labelled neurons with a laser scanner confocal microscope (Leica TCS SP) equipped with a krypton/argon laser. The images were acquired with excitation at 48δnm and they were exported into Photoshop for presentation.

Results

Figure 3 shows confocal images of the IC and the PB to illustrate the result of an injection, in the Ce, of the construct that is under the control of the NSE promoter. As predicted, such injection gave rise to EGFP-labelled neurons in both the IC and the PB (see Figure 3). Figure 4 shows confocal images of the IC and the PB to illustrate the result of an injection, in the Ce, of the construct that is under the control of the 5 kb fragment of the GlyR gene promoter. As hypothesised, such injection gave rise to EGFP-labelled neurons in the PB where GlyR is endogenously expressed but, as predicted, no EGFP-positive neurons were seen in the IC. These results show that the 5 kb fragment of the GlyR gene promoter specifically directs gene expression to a specific sub- population of neurons, i.e., GlyR-expressing neurons.

Example 3 - Sequence Analysis of GlyRalphal promoter

Following sequence assembly, the GLRAl promoter contig was subjected to computational analysis. The programs Proscan vl.7, Promoter 2.0 and Promoter Inspector were used for sequence analysis of the promoter. For comparative purposes, the 1.8 kb promoter sequence from the rat NSE gene, 1 kb of mouse gephyrin promoter - calculated from the ΑTG', 759 bp of the GABA_AR αl gene promoter (accession nos. S693348 and NM_00δ06), 130δ bp of the mouse GlyR β gene promoter (accession no. AJ300577) and 70δ bp from the hGlyR α2 gene (accession no U77724) were also analysed.

Much of the human genome contains repetitive elements, and these retroelements. can be eliminated from the computational promoter analysis using the repeat masker program. The human GlyRalphal sequence shown in SEQ ID No. 1 was therefore analysed to identify the presence of repetitive sequences. The results of that analysis are set out below in Tables 3 and 4. The repeat masker program identified 12 retroelements within the 5723 bp GLRAl sequence. There were also two simple and two low complexity repeats. In total they comprised 1423 bp (24.δ6 %) of the sequence analysed (Table 3).

A complete alignment of the 5723 bp GLRAl promoter fragment, with the same sized promoter fragment from the mouse Glrαl gene was performed using the

CLUSTAL command in the MacVector program. The sequences were aligned from the translational start sites and were found to have approximately 50% sequence identity. The alignment revealed that the larger blocks of sequence identity were between the ΑTG' translational start site and 1270 bp upstream. Alignment of the two 1270 bp fragments showed that they have 74% identity.

Due to the size restrictions of some of the programs used for identification of transcription factors, the previously published 1.1 kb GLRAl promoter fragment was scanned for transcription factor binding sites using Sigscan, Alibaba v2.0, Match and Patch 2.3a and Matlnspector professional release 5.2. Alibaba identified 93 segments as potential transcription factor binding sites, and Transfac identified 266. AliBaba located a number of Spl sites, several AP-2 sites, 2 Oct-1 sites, 10 CCAAT enhancer binding protein domains, and 2 GATA-1 sequences. The TATA box was recognised as a TFDTJ binding site, and the CCAAT box was located at -125. There was also a cAMP response element binding site (CREB) at -395. Patch identified a number of transcription factor elements found in other neuronal genes. These included the mouse PERI 2, human NPY, rat POMC, human neu and mouse neural cell adhesion molecule (NCAM) promoter elements. Match identified 23 sites, which included a neurone-restrictive silencing factor (NRSF) that again, was completely conserved in the mouse. Matlnspector identified three NRSF sites and a CREB site, although the CREB site was in a different position to that identified by AliBaba. Elements with a 5 bp consensus are predicted to occur at least once by chance in a sequence of similar size, so were not subject to further investigation.

Example 4 - Functional Analysis of GlyRalphal promoter

Subcloning the GLRAl Promoter

The 5 'UTR and total GLRAl promoter sequence shown as SEQ ID No. 1 was subcloned as three promoter fragments (1.1, 3.5 and 5.7 kb). Primers 5' GlyRP2FEco and 3' GlyRPlRevHind were designed to amplify the 1.1 kb region, excluding the 5' UTR already contained in pCis-hGlyRαl, with the addition of restriction sites for cloning (Table 1). Human DNA extracted from blood was used as a template in the PCR. The resulting PCR fragment was digested with EcoRl and Hindm. The hGlyRαl cDNA was amplified from pCis-hGlyRαl with 5' GlyRPδHind and 3' GlyRP6Eco primers containing restriction sites for cloning (Table 1). The resulting 1745 bp PCR fragment, containing the entire open reading frame, 2δ7 bp of 5'UTR and 115 bp of 3'UTR, was digested with EcoRl and HindllT and ligated with the 793 bp EcoRl-Hindlll GlyR promoter fragment. Ligation products were separated on a 0.8% lx TAΕ agarose gel. Products of the correct size were excised, purified and cloned into the EcoRl site in pIX.

The 3.5 and 5.7 kb promoter fragments were amplified from the cosmid with the addition of restriction enzyme sites. The 3.5 kb GLRAl sequence was cloned into derivatives of the pIRΕS-ΕGFP vector. For the 3.5 kb pIRΕS-ΕGFP promoter construct, 3205 bp of promoter sequence was PCR amplified from the cosmid with the addition of a Notl site at the 5' end and a Kpnl site at the 3' end (GlyRPδNotF & GlyRPlOKpnR, Table 1). The hGlyR αl cDNA was amplified from pCis-hGlyRαl with primers containing Notl and .Kpnl restriction sites for cloning (GlyRP7NotF & GlyRP9KpnR, Table 1). The resulting PCR fragments were digested with Notl and Kpnl and ligated together. Ligation products were separated on a 0.δ% lx TAΕ agarose gel. Products of the correct size were excised, purified and cloned into the Notl site of a promoterless pIRΕS-ΕGFP plasmid.

In addition to the GLRAl IRΕS-ΕGFP constructs, ΕGFP-fusion constructs were made with all three (1.1, 3.5, 5.7 kb) of the promoter fragments. The fusion constructs were cloned into a promoter-less pcDNA3.1 (-) plasmid (Invitrogen Corp.), where the wild type hGlyRαl cDNA was fused with the ΕGFP gene. This design was based on the N-terminal ΕGFP-GlyR αl fusion described by David-Watine et al., 1999, Neuropharmacology 3δ(6): 7δ5-92.

In vitro promoter function

A neuronal (SK-N-MC) and a non-neuronal (HΕK-293) cell line were transfected with IRES-EGFP and fusion constructs containing the various GLRAl promoter fragments. All transfections utilised the Lipofectamine 2000 reagent. Two days after transfection, cells were assessed for transfection efficiency. Transfection efficiency was estimated to be higher than 90% in the control transfection, and there was little cytotoxicity. However, only the 1,1 kb GLRAl promoter was capable of driving reporter gene expression in the cell lines (data not shown). The 1.1 kb fusion construct gave a stronger signal than the IRES-EGFP constructs - whose signal levels were too low to capture digitally, indicating that the IRES sequence is sub-optimal. As expression of the GLRAl gene is subneuronal, i.e. largely restricted to motor neurones, the only way to test the function of the other promoter fragments is in vivo.

In Vivo Promoter Function

As described above, a tongue injection system was designed to deliver transgene constructs directly to the hypoglossal nucleus. Injection of plasmid DNA into the tongue was followed by gene expression analysis in brainstem sections containing the hypoglossal nucleus.

The tongue injection system was used to test the function of the GLRAl promoter fragments. Injections of IRES-EGFP plasmids carrying 1.1 kb and 3.5 kb of the hGlyRαl promoter, both resulted in EGFP-positive motor neurones in the hypoglossal nucleus six days after injection (data not shown).

Time course of expression in hypoglossal motor neurons.

Constructs were injected into the tongue of mice as described above, except that 20 μl of DNA in 10 μg of distilled water was injected.

At various time points post-injection, animals were sacrificed and the brainstem dissected out, post-fixed and sectioned in 50 micron-thick coronal sections with a vibratome. The sections were mounted on microscope slides and analysed as described above.

The results show that the expression of EGFP protein in the hypoglossal nucleus under the control of the 5.7 kb GlyRalphal promoter sequence after injection into the tongue musculature began at 4 days post injection, peaked at days 5-6 and persisted until day 14.

Discussion Somatic gene delivery is a valuable tool that can be used to deliver foreign genes to the CNS. To gain access to motor neurones, a simple approach is to inject naked/pure DNA into muscle and utilise the natural retrograde processes for its transfer from the tip of the axons innervating the muscle, to the corresponding nerve cell bodies. In the present chapter, a tongue injection system was used to deliver and express transgene constructs in the mouse hypoglossal nucleus. The distribution and expression of the GlyR αl gene is very restricted, which makes its promoter difficult to study in vitro. The development of the tongue injection system made an in vivo analysis possible. Several GLRAl promoter fragments of differing size were isolated and tested for their ability to drive reporter gene expression.

Tongue injections of GLRAl Promoter Fragments

As a mouse tongue is a very small piece of muscle, we had to minimise the volumes of injected DNA solution, so that physical damage and inflammation would be minimal. In addition, too concentrated a DNA solution could lead to increased viscosity and difficulty in injecting the solution into the tongue. Due to these constraints, 5-10 μg of circular plasmid DNA was injected in δ μl of buffer split between four sites on the animal's tongue. As injection proficiency increased, it became apparent that one midline injection of DNA in the tongue was sufficient to produce the widespread reporter gene expression in the hypoglossal nucleus. In addition, the use of microinjection buffer, lx PBS - with or without glucose, or normal saline solution as a diluent did not, at a gross level, affect expression.

The tongue injection system was used to show that the 1.1 kb and 3.5 kb and 5.7 kb GLRAl promoter fragments were able to drive EGFP expression in the hypoglossal nucleus. Neuronal Gene Regulation

Normal functioning of the nervous system relies on the neurones' ability to regulate the expression of specific neurotransmitter receptors. Expression of neural-specific genes may be pan-neuronal or sub-neuronal and have inducible qualities. However, very little is known about the DNA elements and the mechanisms that underlie this regulation.

It has been hypothesised that neuronal gene expression can be achieved by one of four principles, which may operate alone or in combination. The first is that neurone-specific expression is conferred by a neurone-specific basal promoter. In this case, most of the 5' flanking region can be deleted without affecting the specificity of gene expression e.g. 255 bp of the rat NSE promoter can confer cell type specificity.

The second principle is that a relatively non-specific/promiscuous promoter would be switched off in non-neuronal cells e.g. the rat type II sodium channel promoter contains a number of negative regulatory elements like NRSE/NRSF (neuronal restrictive silencer element/factor) that prevents expression in non- neuronal cells.

The third modus operand! is that of a neurone-specific positive modulator (enhancer) that would function in neuronal cells and upregulate transcription from a constitutive minimal promoter.

The fourth means of restricting gene expression is via a promoter that contains both a neural-specific basal promoter and upstream negative regulatory elements that restrict expression to certain subneuronal cell types.

To date, the majority of neural-specific genes that have been studied appear to use negative regulatory mechanisms to direct sub-neuronal specific expression. The presence of the three putative neural restrictive silencing factors (NRSF) elements in the GLRAl promoter, one of which is completely conserved in the mouse GlyR αl promoter, suggests that this gene also uses a negative regulatory mechanism to control its expression. This hypothesis was tested in vitro by transfecting a human neuronal (SK-N-MC) and a non-neuronal cell line (HEK 293) with all three of the GLRAl promoter constructs. Promoter function, assessed by EGFP detection, revealed that only the 1.1 kb GLRAl promoter resulted in EGFP expression in the cell lines.

Early in development, GlyRs are composed of α2 subunit homomers. They are then replaced by the GlyR αl and β subunits that coassemble to form the heteromeric GlyR, found in adult mice and post-natal humans. Unlike GlyR αl, the GlyR β subunit is expressed from birth and is found in areas of the brain where GlyR αl gene expression has not been detected. The variation in spatial and temporal expression of a gene are reflected in the promoter.

Computational analysis of the GLRAl promoter has revealed some interesting possibilities for the regulation of its gene expression. Gel shift assays could be used to examine the capacity of the DNA elements to bind transcription factors, following which, the presence of the irαns-activating factors in the cells of interest can be confirmed. Next, the role of these putative transcription factor elements in GLRAl regulation may be tested by deletion, rearrangement and nucleotide substitution of the promoter sequence. To examine the importance of sub-neuronal specific elements, an in vivo system that utilises retrograde transfer of naked DNA from a discrete brain nucleus could be used, such as the tongue injection system described herein.

Specificity of the GLRAl promoter

A model was developed based on a non-viral, direct brain injection method to evaluate the specificity of the GLRAlpromoter. A system based on the distinctive connectivity of the central nucleus of the amygdala was designed. Figure 2 is a diagrammatic representation of the connectivity of the central nucleus of the amygdala. The central nucleus of the amygdala receives a projection from both the parabrachial nucleus, a brainstem area in which GlyR are highly expressed, and from the insular cortex where neurones do not express GlyR. It was hypothesised that an injection of the transgene under the control of the NSE promoter, in the central nucleus of the amygdala, would result in the retrograde transport and expression of the transgene in both the insular cortex and the parabrachial nucleus. On the other hand, it was predicted that the injection of the transgene under the control of the hGlyR αl gene promoter, despite being transported to both the insular cortex and the parabrachial nucleus, would only be expressed in the parabrachial nucleus. The NSE or the 5.7 kb hGlyR αl promoter EGFP constructs were injected into the central nucleus of the amygdala in C57BL/6 mice. It was found that by contrast to the NSE promoter (Figure 3) the 5.7 kb hGlyR αl promoter was able to drive EGFP expression in the parabrachial nucleus and not the insular cortex (Figure 4). Thus, in contrast with the 1.1 kb promoter, which has broad expression in neuronal and non-neuronal cells, the 5.7 kb promoter was clearly shown to have additional elements that restrict gene expression and confer the spatial restriction found for the endogenous GLRAl gene.

Similar experiments are performed comparing the 1.1 kb promoter fragment with the 3.5 kb fragment.

In summary, the 5.7 kb hGlyR αl promoter confers the natural restricted spatial expression of this gene. Whether this region also contains the regulatory elements that control the temporal aspect of GlyR αl expression would need to be tested using a transgenic animal model.

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are apparent to those skilled in molecular biology or related fields are intended to be within the scope of the invention. Table 3

Number of Length Percentage elements* occupied of sequence

SINEs: 3 523 bp 9.14 %

ALUs 1 300 bp 5.24 % MIRs 2 223 bp 3.90 Q.

Ό

LINEs: 3 729 bp 12.74^' %

LINEl 0 0 bp 0.00 % LINE2 3 729 bp 12.74 Ό L3/CR1 0 0 bp 0.00 %

LTR elements: 0 0 bp 0.00 % MaLRs 0 0 bp 0.00 Ό ERVL 0 0 bp 0.00 Ό

ERV_classI 0 0 bp 0.00 Ό ERV_classII 0 0 bp 0.00 Q. Ό

DNA elements: 0 0 bp 0.00 Ό

MERl_type 0 0 bp 0.00 % MER2 type 0 0 bp 0.00 Q.

Ό

Total interspersed repeats : 1252 bp 21 . 88

Small RNA: 0 0 bp 0 . 00

Satellites : 0 0 bp 0.00

Simple repeats : 2 110 bp 1.92

Low complexity: 2 61 bp 1.07

N.B. Most repeats fragmented by insertions or deletions have been counted as one element.

GC level: 42.46 % Table 4

SW perc perc perc query position in query matching repeat position in repeat score div. del. ins. sequence begin end (left) repeat class/family begin end (left) ID

205 27.3 0.0 6.1 ϋnnamedSeql 20 85 (5375 ) + L2 LINE/L2 3210 3271 (1)

1907 13.7 3.3 0.0 UnnamedSeql 208 507 (4953 + AluSq SINE/Alu 3 312 (1)

190 35.4 12.5 1.0 ϋnnamedSeql 787 882 (4578 + MIR3 SINE/MIR 15 121 (87)

180 0.0 0.0 0.0 ϋnnamedSeql 1026 1045 (4415 + (A)n Simple repeat 1 20 (0)

253 32.5 1.8 1.8 UnnamedSeql 1427 1540 (3920 C L2 LINE/L2 (0) 3313 3200

251 27.3 13.2 3.3 UnnamedSeql 1615 1735 (3725 + MIR3 SINE/MIR 38 170 (38)

252 21.7 3.3 0.0 UnnamedSeql 1737 1796 (3664 + L2 LINE/L2 2598 2659 (655)

1595 25.4 6.8 2.9 UnnamedSeql 1829 2317 (3143 + L2 LINE/L2 2805 3312 (1)

207 34.4 0.0 0.0 UnnamedSeql 3245 3334 (2126 + (CATATA) n Simple repeat 6 95 (0)

257 23.5 8.8 2.9 UnnamedSeql 3452 3553 (1907' + MIR SINE/MIR 152 259 (3)

26 5.0 0.0 0.0 UnnamedSeql 3560 3599 (1861 + AT rich Low complexity 1 40 (0)

211 26.3 12.9 7.4 UnnamedSeql 4079 4214 (1246 4- MIR3 SINE/MIR 9 149 (67)

21 0.0 0.0 0.0 UnnamedSeql 5364 5384 (76) + AT_rich Low complexity 1 21 (0)

Claims

1. A polynucleotide comprising a nucleotide sequence corresponding to the 5' promoter region of a glycine receptor alphal subunit gene or a fragment of said sequence capable of conferring neuron-specific expression of a heterologous nucleotide sequence operably linked thereto.

2. A polynucleotide comprising a nucleotide sequence as shown in SEQ ID. No. 1, or a homologue or fragment of said sequence capable of conferring neuron-specific expression of a heterologous nucleotide sequence operably linked thereto.

3. A polynucleotide according to claim 1 or claim 2 wherein said neuron- specific expression is motor neuron-specific and/or glycine receptor expressing neuron-specific expression.

4. A polynucleotide according to claim 3 which comprises nucleotides 1 to 5397 of the nucleotide sequence as shown in SEQ ID. No. 1

5. A polynucleotide according to any one of claims 1 to 4 operably linked to a heterologous nucleic acid of interest (NOI) such that the polynucleotide directs expression of the NOI in a neuron.

6. A polynucleotide according to claim 5 wherein said neuron is a motor neuron and/or glycine receptor expressing neuron.

7. A polynucleotide according to claim 5 or claim 6 wherein the NOI encodes a polypeptide of therapeutic use.

δ. A polynucleotide according to claim 5 or claim 6 wherein the NOI encodes a polypeptide which is cytotoxic.

9. A polynucleotide according to any one of claims 5 to 8 for use in delivering the NOI to a mammalian neuron.

10. A polynucleotide according to claim 9 wherein said neuron is a motor neuron and/or glycine receptor expressing neuron.

11. A nucleic acid vector comprising a polynucleotide according to any one of the preceding claims.

12. A pharmaceutical composition comprising a polynucleotide according to any one of claims 5 to 10 or a nucleic acid vector according to claim 11 together with a pharmaceutically acceptable carrier or diluent.

13. A polynucleotide according to any one of claims 5 to 10, a nucleic acid vector according to claim 11 or a pharmaceutical composition according to claim 12 for use in a method of treatment of a human or animal.

14. A polynucleotide, nucleic acid vector or pharmaceutical composition according to claim 13 wherein the polynucleotide, nucleic acid vector o pharmaceutical composition are administered to the human or animal by injection into the muscle of the human or animal such that the polynucleotide or nucleic acid vector are delivered to a neuronal cell of said human or animal by retrograde axonal transport.

15. A polynucleotide, nucleic acid vector or pharmaceutical composition according to claim 13 or claim 14 wherein the human or animal is suffering from a disease of, or injury to, the central nervous system.

16. A method of treatment of a human or animal patient suffering from a disease of, or injury to, the central nervous system, or a disease associated with the central nervous system, which method comprises administering an effective amount of a polynucleotide according to any one of claims 5 to 10, a nucleic acid vector according to claim 11 or a pharmaceutical composition according to claim 12 to the patient in need of such treatment.

17. A method according to claim 16 wherein the polynucleotide, nucleic acid vector, viral vector or pharmaceutical composition are administered to the human or animal by injection into the muscle of the human or animal such that the polynucleotide or nucleic acid vector are delivered to a neuronal cell of said human or animal by retrograde axonal transport.

lδ. A method according to claim 16 or claim 17 wherein the human or animal is suffering from a disease of, or injury to, the central nervous system.

19. A method for determining whether a candidate nucleotide sequence is capable of conferring neuron-specific expression of a nucleic acid sequence of interest (NOI) operably linked thereto which method comprises:

(i) providing a non-viral nucleotide vector comprising said candidate nucleotide sequence operably linked to an NOI;

(ii) administering said vector to the brain or brain stem of. a non- human animal; and

(iii) determining whether said NOI is expressed specifically in neurons in the brain or brain stem of said animal.

20. A method according to claim 19 wherein said neurons in step (iii) are motor neurons and/or glycine receptor expressing neurons.

21. A nucleotide sequence capable of confering neuron-specific expression of a nucleotide sequence of interest operably linked thereto, said sequence obtained by the method of claim 19 or claim 20.

22. A method of delivering a nucleic acid to a neuronal cell by non-viral means which method comprises administering said nucleic acid as a naked nucleic acid substantially free of complexing agents, to muscle tissue such that the nucleic acid is transported to said neuronal cell by retrograde axonal transport.