WO2000034488A1 - Bacterial transcription regulators - Google Patents

Abstract

A nucleic acid vector is provided comprising at least one therapeutic gene operably linked to a bacterial transcription regulator DNA-binding site and, optionally, one or more nucleotide sequences of interest encoding a bacterial transcription regulator for binding to said binding site, wherein said bacterial transcriptional regulator is responsive to oxygen and/or redox levels.

Description

BACTERIAL TRANSCRIPTION REGULATORS

Field of the invention

The invention relates to a method of therapeutic gene delivery; means therefor, including components thereof, which have particular, but not exclusive, application in cancer therapy development.

Background to the invention

Knowledge of the molecular genetics of cancer is expanding rapidly. This has led to the development of various novel therapeutic strategies for cancer. Gene-based therapies currently being tested in clinical trials involve largely the ex vivo or in vivo use of viral or liposomal vectors to deliver genes to tumours for: (i) tumour suppressor gene replacement or oncogene inactivation, (ii) the expression of cytokines/vaccines known to activate or enhance anti-tumour immune mechanisms, (iii) enhanced drug sensitivity (e.g. prodrug delivery or activation); (iv) drug resistance for bone marrow protection from high dose chemotherapy, and (v) inhibitors of tumour angiogenesis (1,2).

One of the most crucial aspects of gene therapy for cancer continues to be the targeting of therapeutic gene expression to solid tumours. In some instances, recombinant viral vectors bearing therapeutic genes have been targeted to specific cell types by the insertion of ligands/antibodies into the viral capsid. However, this approach usually requires the expression of tumour-specific antigens by the malignant cell population; which is only rarely the case. Alternatively, naked or viral-incorporated DNA can be injected directly into the tumour (or at least into the local blood supply) to maximise specificity of delivery and expression at the tumour site (1).

Recently, an alternative, entirely novel approach for targeting therapeutic gene expression to tumours has been designed. This utilises the abnormal physiology that exists in almost all solid tumours, regardless of their origin or location, and uses this tumour-specific condition in order to control the expression of heterologous genes (3,4). Aggressive tumours often have insufficient blood supply, partly because tumour cells grow faster than the endothelial cells that make up the blood vessels, and partly because the newly formed vascular supply is disorganised (5). This results in areas of nutrient deprivation, including regions of both reduced oxygen tension (hypoxia) and glucose. Oxygen electrode measurements of tumours have shown that a significant proportion exhibit readings below 2.5 mmHg (normal tissues ranges from 24 to 66 mmHg) (6). Moreover, hypoxic cells are markedly less sensitive to radio- and chemotherapy, which is why, in part, increased levels of tumour hypoxia correlate with reduced survival in many forms of cancer (6).

Hypoxia is a powerful regulator of gene expression in a wide range of different mammalian cell types (7, 8, 9). It acts by the induction of a transcriptional complex termed hypoxia inducible factor- 1 (HIF-1) (9), with its cognate DNA recognition site, the hypoxia- responsive element (HRE) on various mammalian gene promoters. Dachs et al. (3) have used a multimeric form of the HRE from the mouse phosphoglycerate kinase-1 (PGK-1) gene (10) to control expression of both marker and therapeutic genes by human fibrosarcoma cells in response to hypoxia in vitro and within solid tumours in vivo (3). Alternatively, the fact that marked glucose deprivation is also present in ischaemic areas of tumours can be used to activate heterologous gene expression specifically in tumours. A truncated 632 base pair -sequence of the grp78 gene promoter, known to be activated specifically by glucose deprivation, has also been shown to be capable of driving high level expression of a reporter gene in murine tumours in vivo (11). These studies represent the most relevant prior art in so far as they teach that hypoxia or ischaemia responsive elements can be used, in combination with hypoxia or ischaemia inducible factors, to control therapeutic gene expression.

Alternative approaches have involved the use of bacteria to target solid tumours: in one example, clostridial spores of Clostridia beijerinckii were used to target a chemo therapeutic drug-converting enzyme to solid tumours in mice (12). A strain of S. typhimurium has also been used to deliver a reporter or therapeutic protein under the control of a bacterial promoter, to tumours in mice, resulting in anti-tumour activity in vivo (13). Summary of the invention

It is an object of the present invention to provide an oxygen-responsive gene delivery system for the delivery of therapeutic genes to target cells such that their expression is responsive to cellular oxygen levels, for example during hypoxia, the delivery system being based on the use of bacterial transcriptional regulators that are oxygen responsive and their cognate regulatory elements.

We have now shown that FNR upregulates the expression of a reporter gene in response to anoxia in a strain of Salmonella typhimurium.

We have also shown that oxygen-regulated bacterial transcriptional regulators can be expressed in mammalian cells and that they can bind to their cognate DNA-binding site in the context of a mammalian promoter in vitro.

Accordingly, the present invention provides a therapeutic gene delivery vector comprising at least one selected therapeutic gene operatively linked to a bacterial transcription regulator DNA-binding site and, optionally, at least one gene encoding a bacterial transcription regulator for binding to said binding site, wherein said bacterial transcriptional regulator is responsive to oxygen and/or redox levels.

It is preferred that a single delivery vector will contain both the bacterial transcription regulator and the corresponding DNA-binding site, but if required two separate delivery vectors can be used, one for each element, except where a bacterial vehicle is used in which the bacterial transcription regulator is endogenous

In a preferred embodiment of the invention said binding site is positioned selectively either 5' or 3' of a promoter for said therapeutic gene in order to selectively control the activation or repression of said therapeutic gene expression. In yet a further preferred embodiment of the invention a nuclear localisation signal is provided in said delivery vector and ideally is provided adjacent to said bacterial transcription regulator.

In yet a further preferred embodiment of the invention said bacterial transcription regulator comprises at least one gene encoding FNR and/or FLP and said DNA-binding site comprises a corresponding FNR DNA-binding site and/or FLP DNA-binding site.

In a yet further preferred embodiment of the invention said therapeutic gene is a mammalian gene. More preferably still said delivery vector is adapted in conventional manner, or selected, so as to transfect or transform mammalian cells.

According to a yet further aspect of the invention there is provided a pharmaceutical composition comprising the delivery vector of the invention, optionally, including a formulation suitable for the administration of said composition.

According to yet a further aspect of the invention there is provided a method for treating an individual suffering from a disease comprising administering to said individual the delivery vector of the invention which vector is adapted to deliver a therapeutic agent for use in treating said disease.

Detailed description of the invention

Although in general the techniques mentioned herein are well known in the art, reference may be made in particular to Sambrook et al. (14) and Ausubel et al. (15)

A. Oxygen-responsive bacterial transcriptional regulators

Oxygen-responsive bacterial transcriptional regulators include polypeptides and protein complexes encoded by prokaryotic genomes which bind to regulatory elements in promoter sequences in an oxygen-dependent manner. Known examples include Fumarate Nitrate Reduction regulator (FNR) and FNR-like protein (FLP). Typically, an oxygen-responsive bacterial gene regulator for use according to the present invention comprises one or more Fe-S clusters and/or two cysteine thiols capable of forming a disulphide bridge under oxidising conditions. A bacterial gene regulator according to the present invention will often bind to DNA as a dimer, for example a homodimer.

FNR binds to DNA as a dimer, via a site-specific, DNA-binding domain on each subunit. Each subunit contains an N-terminal oxygen-sensing domain, consisting of a highly labile [4Fe-4S] cluster. The switching mechanism between DNA-binding and non-DNA-binding forms seems to involve the assembly/disassembly and/or modification of the Fe-S clusters (16). In the absence of oxygen, [4Fe-4S]²⁺-FNR binds to specific sites in the promoters of different genes in Escherichia coli, thereby activating or repressing their expression (depending on the position of the FNR response site relative to the transcription 'start site'). The amino acid sequence and nucleotide sequence of FNR is given in SEQ I.D. No. l. It is also available from the public domain databases - see accession no. J01608 (g 146960).

FLP has only recently been identified and characterised. It possesses an analogous DNA- binding domain and functions as a dimeric transcriptional regulator, responding to oxygen and/or redox levels via a novel switching mechanism, that involves the reversible formation of a disulphide bridge between two cysteine thiols. In the presence of oxygen, intra-subunit disulphide bond formation induces a conformational change which facilitates

DNA-binding by homodimeric FLP, to specific sequences, that are distinct from FNR-sites. In the absence of oxygen, the disulphide bonds are reduced and DNA-binding activity is abolished. Like FNR, FLP can activate or repress gene expression depending on the position of the FLP response element. The amino acid sequence and nucleotide sequence of FLP is given in SEQ I.D. No.2. It is also available from the public domain databases - see accession no. D00496 - ORF = BAA00388 (g216761).

The operator sequences for both regulators have been established and the following consensus sequences are disclosed: Consensus sequence for the FLP site : C ^A/_c TGANN NTCA ^T/_GG (17)

Consensus sequence for the FNR site : TTGATNNNNATCAA (18).

Oxygen responsive bacterial regulators for use in the present invention also include homologues of FNR and FLP and derivatives thereof, including fragments, that maintain oxygen-responsive DNA-binding properties.

It will be understood that oxygen responsive bacterial regulators for use in the present invention are not limited to the FNR and FLP sequences shown in the sequence listings but also include homologous sequences obtained from any source, for example related viral/bacterial proteins and synthetic peptides, as well as variants or derivatives thereof.

In the context of the present invention, a homologous sequence is taken to include an amino acid sequence which is at least 60, 70, 80 or 90% identical, preferably at least 95 or 98% identical at the amino acid level over at least 20, 30, 40, 50, 75 or 100 amino acids with at least one of the amino acid sequences as shown in the sequence listing herein. In particular, homology should typically be considered with respect to those regions of the sequence known to be essential for DNA-binding and/or oxygen responsiveness rather than non-essential neighbouring sequences. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.

Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences.

% homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues (for example less than 50 contiguous amino acids).

Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion will cause the following amino acid residues to be put out of alignment, thus potentially resulting in a large reduction in % homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximise local homology.

However, these more complex methods assign "gap penalties" to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible - reflecting higher relatedness between the two compared sequences - will achieve a higher score than one with many gaps. "Affine gap costs" are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example when using the GCG Wisconsin Bestfit package (see below) the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

Calculation of maximum % homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (19). Examples of other software than can perform sequence comparisons include, but are not limited to, the BLAST package (see 15 Chapter 18), FASTA (20) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see 15 pages 7-58 to 7-60). However it is preferred to use the GCG Bestfit program. Although the final % homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs. GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). It is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.

Once the software has produced an optimal alignment, it is possible to calculate % homology, preferably % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.

The terms "variant" or "derivative" in relation to the amino acid sequences of the present invention includes any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) amino acid(s) from or to the sequence providing the resultant amino acid sequence has DNA binding activity, preferably having at least the same activity as the polypeptides presented in the sequence listings (Figures 1 and 2).

The FNR and FLP sequences, as well as those of other bacterial oxygen-responsive regulators may be modified for use in the present invention. Typically, modifications are made that maintain the transcriptional regulatory activity of the sequence. Amino acid substitutions may be made, for example from 1, 2 or 3 to 10, 20 or 30 substitutions provided that the modified sequence retains the transcriptional regulatory activity. Amino acid substitutions may include the use of non-naturally occurring analogues.

Conservative substitutions may be made, for example according to the Table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:

Where prokaryotic cells are used in the therapeutic methods of the invention, the cells may already naturally express an oxygen-responsive transcriptional regulator. However where it is desired to use an oxygen-responsive transcriptional regulator that is heterologous to the cell, or where eukaryotic cells, such as mammalian cells, are used, the oxygen-responsive bacterial transcriptional regulators for use in the present invention will typically be expressed in host cells using polynucleotide constructs encoding said regulators. Thus a polynucleotide for use in the invention comprises a nucleic acid sequence encoding an amino acid sequence of an oxygen-responsive bacterial regulator described above. The nucleotide sequences of FNR and FLP are shown in the sequence listings.

It will be understood by a skilled person that numerous different polynucleotides can encode the same polypeptide as a result of the degeneracy of the genetic code. In addition, it is to be understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides of the invention to reflect the codon usage of any particular host organism in which the polypeptides of the invention are to be expressed.

Polynucleotides for use in 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 in order to enhance the in vivo activity or life span of polynucleotides of the invention.

In addition to the particular nucleotide sequences for FNR and FLP shown in the sequence listings, it is also possible to use variants, homologues or derivatives thereof. "Variant",

"homologue" or "derivative" in relation to the nucleotide sequences of FNR or FLP include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid(s) from or to the sequences providing the resultant nucleotide sequence codes for a polypeptide having oxygen-responsive, transcriptional regulatory activity.

Preferably there is at least 75%, more preferably at least 85%, more preferably at least 90% homology to the sequences shown in the sequence listing herein. More preferably there is at least 95%, more preferably at least 98%, homology. Nucleotide homology comparisons may be conducted as described above for amino acid sequences. A preferred sequence comparison program is the GCG Wisconsin Bestfit program described above. 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.

The present invention also encompasses nucleotide sequences that are capable of hybridising selectively to the sequences presented herein, or any variant, fragment or derivative thereof, or to the complement of any of the above. Nucleotide sequences are preferably at least 15 nucleotides in length, more preferably at least 20, 30, 40, 50 or 100 nucleotides in length.

Polynucleotides for use in the invention capable of selectively hybridising to the nucleotide sequences presented herein, or to their complement, will be generally at least 70%, preferably at least 80 or 90% and more preferably at least 95% or 98% homologous to the corresponding nucleotide sequences presented herein over a region of at least 20, preferably at least 25 or 30, for instance at least 40, 60 or 100 or more contiguous nucleotides. The term "selectively hybridizable" means that the polynucleotide used as a probe is used under conditions where a target polynucleotide of the invention is found to hybridize to the probe at a level significantly above background. The background hybridization 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 non-specific 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.

Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex, as taught in Berger and Kimmel (21), 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 hybridization can be used to identify or detect identical polynucleotide sequences while an intermediate (or low) stringency hybridization 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}).

Where the polynucleotide of the invention is double-stranded, both strands of the duplex, either individually or in combination, are encompassed by the present invention. Where the polynucleotide is single-stranded, it is to be understood that the complementary sequence of that polynucleotide is also included within the scope of the present invention. Polynucleotides which are not 100%) homologous to the sequences of the present invention but fall within the scope of the invention can be obtained in a number of ways. Other variants of the sequences described herein may be obtained for example by probing DNA libraries made from a range of individuals, for example individuals from different populations. In addition, other viral/bacterial homologues may be obtained and such homologues and fragments thereof in general will be capable of selectively hybridising to the sequences shown in the sequence listing herein. Such sequences may be obtained by probing cDNA libraries made from or genomic DNA libraries from other bacterial species, and probing such libraries with probes comprising all or part of the sequences shown in SEQ I.D. No. 1 or SEQ I.D. No. 2 under conditions of medium to high stringency.

Variants and strain/species homologues may also be obtained using degenerate PCR which will use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences within the sequences of the present invention. Conserved sequences can be predicted, for example, by aligning the amino acid sequences from several variants/homologues. Sequence alignments can be performed using computer software known in the art. For example the GCG Wisconsin PileUp program is widely used.

The primers used in degenerate PCR will contain one or more degenerate positions and will be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences.

Alternatively, such polynucleotides may be obtained by site directed mutagenesis of characterised sequences, such as the sequences shown in SEQ I.D. No. 1 or SEQ I.D. No. 2. This may be useful where for example silent codon changes are required to sequences to optimise codon preferences for a particular host cell in which the polynucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites, or to alter the property or function of the polypeptides encoded by the polynucleotides. Homologous sequences may also be obtained by using part or all of the sequences shown in

SEQ I.D. No. 1 or SEQ I.D. No. 2 to search the publicly-available database using programs such as BLAST and psi-BLAST.

Polynucleotides according to the invention which encode oxygen-responsive bacterial transcriptional regulators can be incorporated into a recombinant replicable vector. The vector may be used to replicate the nucleic acid in a compatible host cell.

Preferably, a polynucleotide according to the invention which encodes an oxygen- responsive bacterial transcriptional regulator is present in a vector is operably linked to a control sequence that is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector. The term "operably linked" means that the components described are in a relationship permitting them to function in their intended manner. A regulatory 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 sequences.

The control sequences may be modified, for example by the addition of further transcriptional regulatory elements to make the level of transcription directed by the control sequences more responsive to transcriptional modulators.

Vectors of the invention may be transformed or transfected into a suitable host cell as described below to provide for expression of the regulatory protein. This process may comprise culturing a host cell transformed with an expression vector as described above under conditions to provide for expression by the vector of a coding sequence encoding the protein, and optionally recovering the expressed protein.

The vectors may be for example, plasmid or virus vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter. 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 or transform a host cell.

Control sequences operably linked to sequences encoding the protein of the invention include promoters/enhancers and other expression regulation signals. These control sequences may be selected to be compatible with the host cell for which the expression vector is designed to be used in. The term promoter is well-known in the art and encompasses nucleic acid regions ranging in size and complexity from minimal promoters to promoters including upstream elements and enhancers.

The promoter is typically selected from promoters which are functional in mammalian cells where the transcriptional regulatory protein is to be expressed in a mammalian cell or from prokaryotic promoters where the protein is to be expressed in a prokaryotic cell. The promoter is typically derived from promoter sequences of viral, prokaryotic or eukaryotic genes. For example, it may be a promoter derived from the genome of a cell in which expression is to occur. The promoters may be promoters that function in a ubiquitous manner (such as promoters of α-actin, β-actin, tubulin) or, alternatively, a tissue-specific manner (such as promoters of the genes for pyruvate kinase). They may also be promoters that respond to specific stimuli, for example promoters that bind steroid hormone receptors. Viral promoters may also be used, for example the Moloney murine leukaemia virus long terminal repeat (MMLV LTR) promoter, the rous sarcoma virus (RSV) LTR promoter or the human cytomegalovirus immediate early (CMV IE) promoter.

It may also be advantageous for the promoters to be inducible so that the levels of expression of the transcriptional regulator can be regulated during the life-time of the cell. Inducible means that the levels of expression obtained using the promoter can be regulated.

In addition, any of these promoters may be modified by the addition of further regulatory sequences, for example enhancer sequences. Chimeric promoters may also be used comprising sequence elements from two or more different promoters described above. B. Transcriptional regulatory sequences (bacterial HREs) and NOIs.

An oxygen-responsive bacterial transcriptional regulator for use in the present invention is typically used together with a construct comprising a nucleotide sequence of interest (NOI) operably linked to a regulatory sequence to which the transcriptional regulator is capable of binding and modulating the rate of transcription initiated from the promoter of the regulatory sequence. Such as sequence is termed a bacterial HRE. Two examples of bacterial HREs are given below:

In the case of FLP, the consensus sequence for the FLP binding site is: C ^A/_c TGANNNNTCA ^T/_GG (17)

In the case of FNR, the consensus sequence for the FNR binding site is: TTGATNNNNATCAA (18).

The binding site for the transcriptional regulator is typically part of a larger regulatory element which may include a minimal promoter, upstream regulatory elements and optionally in the case of eukaryotic cells, enhancer sequences (see above).

The additional regulatory sequences may be sequences found in eukaryotic genes. For example, it may be a sequence derived from the genome of a cell in which expression of the NOI is to occur, preferably a tumour. Subject to the over-riding control of the HRE construct of the invention, the additional regulatory sequence may function to confer ubiquitous expression or alternatively tissue-specific expression. It is particularly preferred that additional regulatory sequences are used that are preferentially active in one or more specific cell types - such as any one or more of macrophages, endothelial cells or combinations thereof. Further examples include respiratory airway epithelial cells, hepatocytes, muscle cells, cardiac myocytes, synoviocytes, primary mammary epithelial cells and post-mitotically terminally differentiated non-replicating cells such as macrophages. The regulatory sequences are typically operably linked to a nucleotide sequence (nucleic acid) of interest (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.

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, vasoactive proteins and peptides, anti-viral proteins and ribozymes, and derivatives therof (such as with an associated reporter group).

Suitable NOIs for use in the present invention 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); inhibitors of angiogenesis; 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 stimulate 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).

NOI(s) may be used which encode antisense transcripts or ribozymes which interfere with expression of cellular genes for tumour persistence (for example against aberrant myc transcripts in Burkitts lymphoma or against bcr-abl transcripts in chronic myeloid leukemia). The use of combinations of such NOIs is also envisaged.

For further information on the nature of therapeutic genes see WO95/21927 and WO98/15294, the contents of which are incorporated herein by reference.

Instead of, or as well as, being selectively expressed in target tissues, the NOI or NOIs may encode a pro-drug activating 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.

A pro-drug activating enzyme may be delivered to a tumour site for the treatment of a cancer. 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 vector. 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 Reductase); ganciclovir (with HSV thymidine kinase); mustard pro-drugs with nitroreductase and cyclophosphamide (with P450).

Examples of suitable pro-drug activating 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.

Suitable NOIs for use in the treatment or prevention of ischaemic heart disease include NOIs encoding plasminogen activators. Suitable NOIs for the treatment or prevention of rheumatoid arthritis or cerebral malaria include genes encoding anti-inflammatory proteins, antibodies directed against tumour necrosis factor (TNF) alpha, and anti-adhesion molecules (such as antibody molecules or receptors specific for adhesion molecules).

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. In either event, it is preferred for the NOI expression product to demonstrate a bystander effector or a distant bystander effect; that is the production of the expression product in one cell leading to the killing of additional, related cells, either neighbouring or distant (e.g. metastatic), which possess a common phenotype.

Where macrophages or other haematopoietic cells are used, NOIs may be used which encode, for example, cytokines. These would serve to direct the subsequent differentiation of the haematopoietic stemp cells (HSCs) containing a therapeutic NOI. Suitable cytokines and growth factors include but are not limited to: ApoE, Apo-SAA, BDNF, Cardiotrophin- 1, EGF, ENA-78, Eotaxin, Eotaxin-2, Exodus-2, FGF-acidic, FGF-basic, fibroblast growth factor-10, FLT3 ligand, Fractalkine (CX3C), GDNF, G-CSF, GM-CSF, GF-βl, insulin, IFN-γ, IGF-I, IGF-II, IL-lα, IL-lβ, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (72 a.a.), IL-8 (77 a.a.), IL-9, IL-10, IL-11, IL-12, IL-13,^' IL-15, IL-16, IL-17, IL-18 (IGIF), Inhibin α, Inhibin β, IP- 10, keratinocyte growth factor-2 (KGF-2), KGF, Leptin, LIF, Lymphotactin, Mullerian inhibitory substance, monocyte colony inhibitory factor, monocyte attractant protein, M-CSF, MDC (67 a.a.), MDC (69 a.a.), MCP-1 (MCAF), MCP-2, MCP-3, MCP- 4, MDC (67 a.a.), MDC (69 a.a.), MIG, MlP-lα, MlP-lβ, MIP-3α, MIP-3β, MIP-4, myeloid progenitor inhibitor factor- 1 (MPIF-1), NAP-2, Neurturin, Nerve growth factor, β- NGF, NT-3, NT-4, Oncostatin M, PDGF-AA, PDGF-AB, PDGF-BB, PF-4, RANTES, SDFlα, SDFlβ, SCF, SCGF, stem cell factor (SCF), TARC, TGF-α, TGF-β, TGF-β2, TGF-β3, tumour necrosis factor (TNF), TNF-α, TNF-β, TNIL-1, TPO, VEGF, GCP-2, GRO/MGSA, GRO-β, GRO-γ and HCC1.

For some applications, a combination of some of these cytokines may be preferred, in particular a combination which includes IL-3, IL-6 and SCF, for the maintenance and expansion of stem cell populations. For differentiation in vitro, further cytokines may be added such as GM-CSF and M-CSF to induce differentiation of macrophages or GM-CSF and G-CSF to obtain neutrophils. On reintroduction of the engineered cells into the individual from whom they were derived, the body's own mechanisms then permit the cells or their differentiated progeny to migrate into the affected area e.g. the tumour.

Optionally, another NOI may be a suicide gene, expression of which in the presence of an exogenous substance results in the destruction of the transfected or transduced cell. An example of a suicide gene includes the herpes simplex virus thymidine kinase gene (HSV tk) which can kill infected and bystander cells following treatment with ganciclovir.

Optionally another NOI may be a targeting protein (such as an antibody to the stem cell factor receptor (WO-A-92/17505; WO-A-92/21766)). For example, recombinant (ecotropic) retroviruses displaying an antibody (or growth factor or peptide) against a receptor present on HSCs (CD34 or stem cell factor, for example) might be used for targeted cell delivery to these cells, either ex vivo by incubating unfractionated bone marrow with virus or by intravenous delivery of virus.

NOIs may also include marker genes (for example encoding β-galactosidase or green fluorescent protein) or genes whose products regulate the expression of other genes. 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 β-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.

In a preferred embodiment, at least one NOI, operably linked to a bacterial HRE according to the present invention encodes an oxygen-responsive bacterial transcriptional regulatory protein such as FNR. Such a construct will provide an autoregulated system since in the presence of hypoxia, expression of the bacterial transcriptional regulatory protein from the HRE construct will increase and serve to further increase transcription from the HRE construct and other HRE constructs present.

Nucleotide vectors

Polynucleotides for use in the invention can be incorporated into a recombinant replicable vector. The vector may be used to replicate the nucleic acid in a compatible host cell. The vector may be recovered from the host cell. Suitable host cells include bacteria such as E. coli and S. typhimurium, yeast, mammalian cells and other eukaryotic cells, for example insect Sf9 cells.

A vector comprising a bacterial HRE 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 HRE. 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 according to 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 HRE/promoter sequences to enable an NOI to be cloned into the vector when required whereby it is then operably linked to the HRE/promoter sequences. It is preferred that a single delivery vector will contain both the sequence encoding a bacterial transcription regulator and the corresponding DNA-binding site (bacterial HRE), but if required two separate delivery vectors can be used, one for each element.

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.

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 polylysine, 1, 2,-bis (oleoyloxy)-3- (trimethylammonio) propane (DOTAP)-cholesterol complexes and combinations thereof.

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

The delivery vector systems may be used therapeutically to target a patient's cells directly. However, they may also be used to transform or transfect bacteria such as anaerobic bacteria, and mammalian cellular vectors such as macrophages, that is a plasmid or phage may be used for transforming or transfecting a further cell type that is ideally adapted to target the therapeutic gene to a selected site. Thus it may be used to transform a macrophage. Indeed, in a preferred embodiment of the invention said delivery vector comprises macrophages which advantageously migrate to hypoxic sites and so have a natural tendency to infiltrate hypoxic or ischaemic areas and so deliver therapeutic genes ideally under the control of hypoxia responsive regulators to appropriate target sites.

Alternatively in the instance where the delivery vector is a plasmid or phage it may be suitably placed at a target site, for example, by way of injection or the like, or indeed by way of using any conventional means.

However, as previously mentioned, other delivery vectors may be selected and adapted for targeted delivery by conventional means. For example, viral vectors may be selected and provided with ligands/antibodies in the viral capsid to ensure that the viral vector targets and so binds to a specific tissue type.

D. Host cells and target cells

Polynucleotide constructs, nucleic acid vectors and viral vectors of the invention may be introduced into a variety of host cells. Host cells include both prokaryotic, for example bacterial, and eukaryotic, for example yeast and higher eukaryotic cells (such as insect, mammalian, for example human, cells). Host cells may be used to propagate both non- viral and viral vectors, for example to prepare nucleic acid vectors comprising a polynucleotide of the invention or to prepare high titre viral stocks.

Alternatively, host cells comprising a polynucleotide of the invention/NOI may be used in therapy, such as the use of macrophages discussed below in, for example, ex vivo therapy.

Non-viral nucleic acids and viral vectors are typically introduced into host cells using techniques well known in the art such as transformation or transfection. Viral vectors may also be introduced into host cells by infection.

Target cells, iri the context of the present invention, means cells of, or from, an organism that typically it is desired to treat, rather than simply cell lines. Target cells may be removed from the organism and subsequently returned after treatment, or targeted in vivo.

Thus for example, tumour cells in vivo can be considered to be target cells.

Examples of target cells include tumour cells (see below for list of tumours), in particular. tumour cells under conditions of hypoxia. The target cell may be a growth-arrested cell capable of undergoing cell division such as a cell in a central portion of a solid tumour mass or a stem cell such as an HSC or a CD34⁺ cell. As a further alternative, the target cell may be a precursor of a differentiated cell such as a monocyte precursor, a CD34+ cell, or a myeloid precursor. The target cell may also be a differentiated cell such as a neuron, astrocyte, glial cell, microglial cell, macrophage, monocyte, epithelial cell, endothelial cell or hepatocyte. Target cells may be transfected or transduced either in vitro after isolation from an individual or may be transfected or transduced directly in vivo.

In a particularly preferred embodiment, haematopoietic stem cells such as macrophages are used as host cells/target cells. HSCs are pluripotent stem cells that give rise to all blood cell lineages in mammals. HSCs differentiate into various cell lineages under the influence of microenvironmental factors such as cell-to-cell interactions and the presence of soluble cell cytokines. Four major cell lineages arise from the HSCs. These include: erythroid (erythrocytes); megakaryocytic (platelets); myeloid (granulocytes and monocytes); and lymphoid (lymphocytes). Maturation of these cells occurs under the influence of a network of tissue specific protein regulators which have been given a variety of names including growth factors, cytokines and interleukins.

Macrophages, derived from monocytes from the bloodstream, have been used as a delivery vehicle for targeting drugs and therapeutic genes to solid tumours. It has been shown that macrophages continually enter solid tumours and congregate in poorly vascularised, ischaemic sites in breast carcinomas. Moreover, the degree of ischaemia-induced necrosis in these tumours was positively correlated with the degree of intra-tumoral macrophage infiltration.

Monocytes and macrophages also infiltrate ischaemic lesions which are a feature of other disease states including cerebral malaria, coronary heart disease and rheumatoid arthritis. Thus monocytes and macrophages are suitable host cells for use in the present invention. In particular, monocytes and/or macrophages comprising polynucleotides and/or vectors, such as viral vectors, of the invention are suitable for use in ex vivo and in vivo methods for treating diseases associated with hypoxia.

E. Therapeutic Uses

The selective use, either in isolation or combination, of bacterial transcription regulator(s), enables gene expression to be controlled under a range of oxygen states; moreover, they can be used to control gene expression under a range of oxygen states in mammalian cells, as well as in bacteria. These regulators can therefore, in isolation or combination, be used to drive the expression of therapeutic genes in various oxygen states and thus to treat conditions characterised by a specific oxygen state. For example, without limitation, these regulators can be used to treat diseases/conditions/tissue characterised by hypoxia such as ischaemic heart tissue, central nervous tissue during cerebral malaria, arthritic joints, retinal tissue in diabetic retinopathy, and cancer tissue.

Notably, the binding of FNR or FLP to a specific DNA-binding site brings about activation or repression of gene expression depending on the position of the said binding site relative to the transcription "start site".

Depending upon the nature of the therapeutic gene construct and the relative positioning of the FNR/FLP DNA-binding site vis-a-vis the promoter and/or enhancer sequences of the therapeutic gene it may be possible to control whether the bacterial regulator acts in a positive or a negative way, vis-a-vis gene expression, in response to oxygen and/or redox levels.

It is therefore an object of the invention to provide a novel therapeutic gene expression system which is responsive to oxygen and/or redox levels and which involves the use of prokaryotic transcription regulators to regulate the expression of eukaryotic therapeutic genes, optionally, in prokaryotic or eukaryotic expression systems. It is yet a further object of the invention to provide a novel drug delivery system which is responsive to oxygen levels and which involves the use of a prokaryotic transcriptional regulator and a prokaryotic or a eukaryotic therapeutic gene.

It will be understood, that in one embodiment of the invention a single bacterial transcription regulator will be selected for use in said delivery vector in combination with the selection of the corresponding DNA-binding site. In this embodiment of the invention preferably the selective positioning of the DNA-binding site, vis-a-vis, different therapeutic genes and their promoter may be varied in a single delivery vector. Alternatively, in other embodiments of the invention both of the aforementioned bacterial transcription regulators, and the corresponding DNA-binding sites, may be selected for use in a single delivery vector, and in this embodiment of the invention it is envisaged that, advantageously, each of said bacterial transcription regulators will be selectively positioned vis-a-vis a different therapeutic gene so as to provide for differential gene expression.

In either case, delivery vectors can be used to target therapeutic genes to given tissue and moreover, the expression of said therapeutic genes can be selectively controlled with regard to oxygen and/or redox states. Thus, in one example, a given therapeutic agent encoded by a therapeutic gene will be produced under hypoxic conditions and then, as treatment progresses, and the hypoxic state declines, the emergence of aerobic conditions will result in the expression of a second alternative therapeutic agent encoded by a different therapeutic gene under the control of an aerobic bacterial transcription regulator. This successive nature of therapeutic gene production may occur in the reverse order and may also occur cyclically depending upon oxygen and/or redox levels and indeed fluctuations in oxygen states during the progression of a physiological or diseased condition.

It will be apparent that the response of the delivery vector to a given oxygen state will be determined by the selection of at least one bacterial transcription regulator and the positioning of the corresponding DNA-binding domain with respect to the promoter of the therapeutic gene controlled thereby. The present invention is believed to have a wide therapeutic applicability - depending on inter alia the selection of the one or more NOIs. In addition, or in the alternative, the present invention may be useful in the treatment of disorders listed in WO-A-98/09985. For ease of reference, part of that list is now provided: macrophage inhibitory and/or T cell inhibitory activity and thus, anti-inflammatory activity; anti-immune activity, i.e. inhibitory effects against a cellular and/or humoral immune response, including a response not associated with inflammation; inhibit the ability of macrophages and T cells to adhere to extracellular matrix components and fibronectin, as well as up-regulated fas receptor expression in T cells; inhibit unwanted immune reactions and inflammation including arthritis, including rheumatoid arthritis, inflammation associated with hypersensitivity, allergic reactions, asthma, systemic lupus erythematosus, collagen diseases and other autoimmune diseases, inflammation associated with atherosclerosis, arteriosclerosis, atherosclerotic heart disease, reperfusion injury, cardiac arrest, myocardial infarction, vascular inflammatory disorders, respiratory distress syndrome or other cardiopulmonary diseases, inflammation associated with peptic ulcer, ulcerative colitis and other diseases of the gastrointestinal tract, hepatic fibrosis, liver cirrhosis or other hepatic diseases, thyroiditis or other glandular diseases, glomerulonephritis or other renal and urologic diseases, otitis or other oto-rhino-laryngological diseases, dermatitis or other dermal diseases, periodontal diseases or other dental diseases, orchitis or epididimo-orchitis, infertility, orchidal trauma or other immune-related testicular diseases, placental dysfunction, placental insufficiency, habitual abortion, eclampsia, pre-eclampsia and other immune and/or inflammatory-related gynaecological diseases, posterior uveitis, intermediate uveitis, anterior uveitis, conjunctivitis, chorioretinitis, uveoretinitis, optic neuritis, intraocular inflammation, e.g. retinitis or cystoid macular oedema, sympathetic ophthalmia, scleritis, retinitis pigmentosa, immune and inflammatory components of degenerative fondus disease, inflammatory components of ocular trauma, ocular inflammation caused by infection, proliferative vitreo-retinopathies, acute ischaemic optic neuropathy, excessive scarring, e.g. following glaucoma filtration operation, immune and/or inflammation reaction against ocular implants and other immune and inflammatory- related ophthalmic diseases, inflammation associated with autoimmune diseases or conditions or disorders where, both in the central nervous system (CNS) or in any other organ, immune and/or inflammation suppression would be beneficial, Parkinson's disease, complication and/or side effects from treatment of Parkinson's disease, AIDS-related dementia complex HIV-related encephalopathy, Devic's disease, Sydenham chorea, Alzheimer's disease and other degenerative diseases, conditions or disorders of the CNS, inflammatory components of stokes, post-polio syndrome, immune and inflammatory components of psychiatric disorders, myelitis, encephalitis, subacute sclerosing pan- encephalitis, encephalomyelitis, acute neuropathy, subacute neuropathy, chronic neuropathy, Guillaim-Barre syndrome, Sydenham chora, myasthenia gravis, pseudo-tumour cerebri, Down's Syndrome, Huntington's disease, amyotrophic lateral sclerosis, inflammatory components of CNS compression or CNS trauma or infections of the CNS, inflammatory components of muscular atrophies and dystrophies, and immune and inflammatory related diseases, conditions or disorders of the central and peripheral nervous systems, post-traumatic inflammation, septic shock, infectious diseases, inflammatory complications or side effects of surgery, bone marrow transplantation or other transplantation complications and/or side effects, inflammatory and/or immune complications and side effects of gene therapy, e.g. due to infection with a viral carrier, or inflammation associated with AIDS, to suppress or inhibit a humoral and/or cellular immune response, to treat or ameliorate monocyte or leukocyte proliferative diseases, e.g. leukaemia, by reducing the amount of monocytes or lymphocytes, for the prevention and/or treatment of graft rejection in cases of transplantation of natural or artificial cells, tissue and organs such as cornea, bone marrow, organs, lenses, pacemakers, natural or artificial skin tissue.

In particular, polynucleotides, nucleic acid vectors, viral vectors and host cells of the present invention may be used in the treatment of tumours. Examples of tumours that may be treated by the present invention include but are not limited to: sarcomas including osteogenic and soft tissue sarcomas, carcinomas such as breast, lung, bladder, thyroid, prostate, colon, rectum, pancreas, stomach, liver, uterine, and ovarian carcinoma, lymphomas including Hodgkin and non-Hodgkin lymphomas, neuroblastoma, melanoma, myeloma, Wilms tumour, and leukemias, including acute lymphoblastic leukemia and acute myeloblastic leukemia, gliomas and retinblastomas. A particularly advantageous feature of the present invention is that NOIs may be expressed in hypoxic cells and not cells under normoxic conditions. Thus polynucleotides, nucleic acid vectors, viral vectors and host cells of the present invention may be used in the clinical management of a range of conditions characterised by hypoxia. Example of conditions which are characterised by symptoms of hypoxia include stroke, deep vein thrombosis, pulmonary embolus and renal failure. The cell death of cardiac tissue, called myocardial infarction, is due in large part to tissue damage caused by ischemia and/or ischemia followed by reperfusion. Other examples include cerebral malaria and rheumatoid arthritis. It is especially preferred to use polynucleotides, nucleic acid vectors, viral vectors and host cells of the present invention in the clinical management of solid tumours such as ovarian tumours, in particular tumours comprising tumour cells under hypoxic conditions. Treatment may effect a slowdown in the rate of tumour growth, a cessation in the rate of tumour growth or indeed shrinkage of tumour mass without necessarily resulting in complete apoptotic/necrotic death of all malignant cells in an affected patient.

The polynucleotides, nucleic acid vectors, viral vectors and host cells of the present invention may also be used in preventative medicine. Thus the NOIs used in the invention may have a therapeutic effect via prophylaxis. For example, where an increased risk of developing cancer is diagnosed, the invention may be used to vaccinate the at-risk individual.

Suitability for prophylaxis may be based on genetic predisposition to cancer, for example cancer of the breast or ovary because of one or more mutations in a BRCA-1 gene, a BRCA-2 gene (22) or another relevant gene.

G. Administration

The polynucleotide, nucleic acid vectors and viral vectors of the invention may thus be used to deliver therapeutic genes to a human or animal in need of treatment.

The polynucleotide of the invention may be administered directly as a naked nucleic acid construct, preferably further comprising flanking sequences homologous to the host cell genome. Uptake of naked nucleic acid constructs by mammalian cells is enhanced by several known techniques including biolistic transformation and lipofection. Alternatively, the polynucleotide may be administered as part of a nucleic acid vector, including a plasmid vector or viral vector, preferably a lentiviral vector.

Preferably the delivery vehicle (i.e. naked nucleic acid construct or viral vector comprising the polynucleotide for example) is combined with a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical composition. Thus, the present invention also provides a pharmaceutical composition for treating an individual by gene therapy, wherein the composition comprises a therapeutically effective amount of the polynucleotide, vector or viral vector of the present invention comprising one or more deliverable therapeutic and/or diagnostic NOI(s) or a viral particle produced by or obtained from same, together with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant. The pharmaceutical composition may be for human or animal usage.

The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. Suitable carriers and diluents include isotonic saline solutions, for example phosphate-buffered saline. The pharmaceutical compositions may comprise as - or in addition to - the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s), and other carrier agents that may aid or increase the viral entry into the target site (such as for example a lipid delivery system).

The pharmaceutical composition may be formulated for parenteral, intramuscular, intravenous, intracranial, subcutaneous, intraocular or transdermal administration or inhalation.

Where appropriate, the pharmaceutical compositions can be administered by any one or more of: inhalation, in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment or dusting powder, by use of a skin patch, orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents, or they can be injected parenterally, for example intracavernosally, intravenously, intramuscularly or subcutaneously. For parenteral administration, the compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood. For buccal or sublingual administration the compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.

The pharmaceutical composition is administered in such a way that the polynucleotide/vector containing the therapeutic gene for gene therapy, can be incorporated into cells at an appropriate area.

When the polynucleotide of the invention is delivered to cells by a viral vector of the invention, the amount of virus administered is in the range of from 10³ to 10¹⁰ pfu, preferably from 10 to 10 pfu, more preferably from 10 to 10 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 NOI is under the control of an inducible regulatory sequence, 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 is stopped. 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 tissue-specific promoters will be of assistance in the treatment of disease using the polynucleotides/vectors of the invention. 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.

Host cells, such as prokaryotic cells or mammalian cells, of the invention may be administered to a patient or an at-risk individual in a suitable formulation. The formulation may include an isotonic saline solution, a buffered saline solution or a tissue-culture medium. The cells are administered by bolus injection or by infusion intravenously or directly to the site of a tumour or to the bone marrow at a concentration of for example between approximately 1

or 10¹⁰ cells per dose. Prokaryotic cells are typically grown to mid-log phase, harvested by low speed centrifugation and resuspended in a suitable buffer such as 5 % sodium bicarbonate to a final volume of, for example, 10 to 100 μl (see 23), prior to administration.

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.

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 - Graph showing oxygen- and/or nitrate-regulated expression of lacZ by FNR in S. typhimurium (LB5010), via a single FNR-binding site in an E. coli nitrate-independent (pGS810) or nitrate-dependent (pnir ) promoter

Figure 2 - Western blot of extracts from HT1080 cells transfected with pcI-NeoNflp and treated as follows - 1 : normoxia, 2: normoxia + 4hr ImM H₂O₂, 3: normoxia + 6hr 0.75mM H₂O₂, 4: anoxia 16 hr, 5: untransfected HT1080 cells, 6: positive control FLP, oxidised, 7: positive control FLP, reduced.

Figure 3 - shows a Western blot of extracts from 293 cells transfected with pclneo-fnr, without or with pAdVantage: lanes 1 + 2: duplicate transfections with pclneo-fnr alone, lanes 3 + 4: duplicate transfections with pclneo-fnr + pAdVantage lane 5: untransfected cells, lane 6: positive control extract

Figure 4 - Gel retardation of Xhol-Hinάlll fragment of pGL3 -control containing a single FNR- or FLP-binding site near the TATA box, by FLP purified from E. coll

Figure 5 - shows an example of an FLP/FNR mammalian expression plasmid with (A) or without (B) a nuclear localisation signal.

Figure 6 - shows repressor reporter plasmids, with FNR/FLP-binding site downstream of transcription initiation site (at Hind III site), or nearer TATA box of SV40 promoter (small letters: sequences from vector, capital letters: inserted DNA, bold letters: most conserved bases in consensus sequence). FNR and FLP binding sites were designed based on consensus sequence for each (18, 17).

A. shows an example of an FNR mammalian reporter plasmid (repressor).

B. shows an example of an FLP reporter plasmid (repressor).

Figure 7 - shows an activator reporter plasmid, with FNR/FLP-binding site upstream of SV40 promoter (small letters: sequences from vector, capital letters: inserted DNA, bold letters: most conserved bases in consensus sequence).

Figure 8 - shows activator reporter plasmids, in which a bacterial promoter containing one or multiple FNR-binding sites directs the expression of a reporter (lac Z) (bold: most conserved bases). Sequences were determined by Bell et al. (24) (FF/ne/R) and Jayaraman P-S et al (25) (pnirB). SEQ I.D. No. 1 - shows the nucleotide and amino acid sequence of the fnr gene and the encoded protein. The amino acid sequence of FNR is given below the nucleotide sequence which encodes it. The -10 and -35 regions are overlined and the FNR-sites are in bold. The Hind III, Eco l, and BamHl restrictions sites are underlined.

SEQ I.D. No. 2 - shows the nucleotide and amino acid sequence of the flp gene. Both strands of the flp gene and surrounding sequences are shown. The amino acid sequence of the FLP protein is given below the nucleotide sequence in capitals and the other possible translational start sites are indicated with arrows and the extra amino acids derived from these start site are given in lower case. The -10, -35 and Shine-Dalgarno sequences are shown in bold. The primers, FLP1 and FLP2, used to create the Ncol and Sail restriction sites necessary for cloning are shown, with the non-complementary nucleotides in bold. Restriction sites for Hindlll, Ncol, EcoRl, Sail and Kpnl are underlined. An inverted repeat region, probably constituting the termination signal, is also marked (^Λ).

EXAMPLES

Example 1 - Use of FΝR/FLP to Activate Gene Expression in Bacteria

General principles

1. Typically, bacterial strains are chosen such that FΝR/FLP is expressed endogenously and does not need to be expressed from a plasmid.

2. The reporter plasmid is based on pRW50 (19), in which a promoter containing a single or multiple FΝR FLP -binding sites is introduced between the EcoRI and H dIII sites, and directs the expression of a reporter gene (lac Z) or therapeutic gene (Figure 8).

Expression will be activated by FΝR in the absence of oxygen only and by FLP in the presence of oxygen only. Experimental

Salmonella typhimurium LB5010 transformed with pGS810 (pRW50 with FFwe R promoter cloned into EcoRI-BamHl site - see Figure 8) or pnirB (pRW50 with nirB promoter cloned into EcoKL-BamHI site - see Figure 8) were cultured under aerobic or anaerobic conditions or in the presence of nitrate, and lacZ expression was measured from duplicate cultures, using the standard beta-galactosidase assays (values for duplicates were within 11%) or less). The results shown in Figure 1 demonstrate oxygen- and/or nitrate- regulated expression of lacZ by FNR

Replacement of the lacZ gene with a therapeutic gene will enable prokaryotes such as attenuated strains of bacteria transformed with constructs of the invention to be used as whole cell vectors for delivery therapeutic gene products to animal or human patients.

Example 2 - Expression of FLP and FNR in mammalian cells

A, FLP

HT1080 cells were transfected with the mammalian expression plasmid pCIneo-Nflp (see

Figure 5 and Example 3) which encodes NFLP and pAdVantage, using the FuGene 6 reagent (Boehringer Mannheim), according to the protocol optimised by Oxford BioMedica. Cells were treated as described in the figure legend to Figure 2 and extracts were prepared 24 hours post-transfection using Passive Lysis Buffer (Dual-Luciferase Reporter Kit, Promega) as recommended by manufacturers. Extracts were analysed by non- reducing SDS-PAGE (denaturing polyacrylamide gel electrophoresis) in order to visualise the balance of oxidised versus reduced forms of NFLP, and Western Blotting.

The results shown in Figure 2 demonstrate that in HT1080 cells, NFLP is expressed and found mainly in its reduced form, but it can be converted to the oxidised form by addition of hydrogen peroxide (4hr 1 mM, or 6hr 0.75 mM) in the medium (foetal calf serum-free) - see Figure 2 R FNR

293 cells were transfected with the mammalian expression plasmid pCIneo-fnr (see Figure

5 and Example 3), which encodes FNR, with or without pAdVantage (Promega), using the

FuGene 6 reagent (Boehringer Mannheim), according to the protocol optimised by Oxford BioMedica. Extracts were prepared as described above and analysed by reducing SDS-

PAGE and Western Blotting.

The results shown in Figure 3 demonstrate that in 293 cells, FNR is expressed, and expression is enhanced in the presence of pAdVantage.

These results therefore demonstrate that active FNR and FLP that is sensitive to oxygen status may be expressed in mammalian cells.

Example 3 - FLP binds to FLP sites but not FNR sites in a mammalian expression vector

FLP was overexpressed in, and purified from, E. coli and then incubated with a radiolabelled Xhol-Hinάlll fragment of pGL3 -control (Promega) containing a single FNR- or FLP -binding site near the TATA box. DNA-protein complexes were analysed by non- denaturing PAGE and autoradiography.

The results shown Figure 4 demonstrate that m vitro, E. coli purified FLP binds to a DNA fragment containing the FLP binding site in the context of the pGL3 -control promoter but not to a similar fragment containing the FNR binding site in the same position and therefore that protein and site-specific binding can be achieved using mammalian vectors.

Example 4 - Regulation of gene expression in mammalian cells

General principles

^' (i) Use of FLP or FNR to Repress Mammalian Gene Expression in mammalian cells 1. A suitable expression plasmid may be based on pCIneo (Promega). The flp or fnr coding region, as appropriate, are introduced downstream of the CMV enhancer-promoter sequence into the polylinker, and fused at the 5' end to a consensus Kosak sequence (26) for efficient translation, and optionally the adenoviral El A nuclear localisation signal (NLS) (Figures 5a and b).

2. The reporter plasmids may be based on pGL3-control (Promega) in which one or multiple copies of the FLP- or FNR-binding site(s) is/are introduced in or near the 3 ' end of SV40 early promoter and enhancer sequences directing the expression of a reporter gene (e.g. lacZ, cat, gfp or luciferase) or a therapeutic gene (e.g. the sequence encoding the soluble domain of the human VEGF receptor, flk-l or flt-l which will block tumour angiogenesis) (Figure 6a and b). This design was based on work involving cAMP -receptor protein (CRP) (27), a member of the FNR family of proteins which is responsive to cAMP.

Expression of the reporter gene or the therapeutic gene will be repressed by the FLP in the presence of oxygen, and de-repressed in the absence of oxygen whereas FNR represses their expression in the absence of oxygen.

(ii) Use of FLP or FNR to Activate Mammalian Gene Expression

A vector has been used previously to activate gene expression in response to hypoxia, using the hypoxia-response element (HRE) in mammalian cells. This vector provides a good basis for the construction of an FNR/FLP -regulated equivalent.

1. The expression plasmid is identical to that indicated above in section (i) part 1.

2. The reporter plasmids are based on pGL3pro (Promega) in which one or multiple copies of FLP- or FNR-binding site(s) is/are introduced at the Bglll site upstream of the SV40 minimal promoter (see Figure 7) directing the expression of a reporter gene (Luciferase) or a therapeutic gene (e.g. the sequence encoding the soluble domain of the human VEGF receptor, flk-l or flt-l) (Figure 7). Expression of the reporter gene or the therapeutic gene here will be activated by FLP in the presence of oxygen only, and activated by FNR in the absence of oxygen only.

Variations on this theme could include making hybrid DNA-binding proteins, which incorporate combinations of mammalian and prokaryotic DNA-binding, redox/oxygen- sensing and RNA polymerase activating domains. For example the DNA-binding domain of HIF or other mammalian transcription factors could be used with the oxygen-sensing domain of FLP or FNR.

Delivery Vehicle for DNA Constructs

These constructs could be inserted into conventional viral or non-viral vectors for delivery to the diseased site (e.g. tumour), or transported by novel cellular vectors such as macrophages which have been shown to target hypoxic sites in diseased tissues. Alternatively, non-pathogenic anaerobic or facultative anaerobic bacteria such as Clostridia beijerinckii or S. typhimurium could be used for transporting an FLP/FNR-regulated therapeutic gene (in which case, bacterial expression and/or reporter vectors would be used in place of the mammalian ones indicated above, to introduce the FLP/FNR gene and response elements into these cells).

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 obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims. REFERENCES

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Claims

1. A nucleic acid vector comprising at least one therapeutic gene operably linked to a bacterial transcription regulator DNA-binding site and, optionally, one or more nucleotide sequences of interest encoding a bacterial transcription regulator for binding to said binding site, wherein said bacterial transcriptional regulator is responsive to oxygen and/or redox levels.

2. A vector according to claim 1 wherein the bacterial transcription regulator(s) is/are selected from fumarate nitrate reduction regulator (FNR) and/or a FNR-like protein (FLP) and said DNA-binding site comprises a corresponding FNR DNA-binding site and/or FLP DNA-binding site.

3. A vector according to claim 1 or 2 wherein the binding site is positioned selectively either 5' or 3' of a promoter for said therapeutic gene in order to selectively control the repression or activation of said therapeutic gene expression.

4. A vector according to any one of claims 1 to 3 wherein the therapeutic gene is a mammalian gene.

5. A vector according to any one of claims 1 to 4 which is capable of being transfected or transformed into mammalian cells.

6. A viral vector comprising a vector according to any one of the preceding claims.

7. A viral vector according to claim 6 which selected from a retroviral vector and an adenoviral vector.

8. A host cell comprising a vector according to any one of claims 1 to 7.

9. A host cell according to claim 8 which further comprises a nucleotide sequence of interest capable of expressing a bacterial transcription regulator polypeptide which polypeptide is capable of binding to the binding site present in the nucleic acid vector.

10. A host cell according to claims 8 or 9 selected from a prokaryotic cell and a mammalian cell.

1 1. A pharmaceutical composition comprising a nucleic acid vector according to any one of claims 1 to 7 together with a pharmaceutically acceptable carrier or diluent.

12. A pharmaceutical composition comprising a host cell according to any one of claims 8 to 10 together with together with a pharmaceutically acceptable carrier or diluent.

13. A vector according to any one of claims 1 to 7 or a host cell according to any one of claims 8 to 10 for use in treating an individual suffering from a disease.