WO2005052164A1 - Gene therapy - Google Patents

Abstract

A vector system is provided which comprises a first component (a) comprising a first promoter element composed of a tetracycline-regulated promoter upstream of a nucleic acid sequence encoding a first protein in which said nucleic acid sequence is upstream of a termination of transcription signal; and a second component (b) comprising a second promoter element upstream of a nucleic acid sequence encoding the reverse tetracycline transactivator (rtTA) protein rtTA2S-M2 which is upstream of an internal ribosomal entry site (IRES) which is upstream of a nucleic acid sequence which encodes the tetracycline repressor protein tetR-KRAB, which is upstream of a termination of transcription signal in which the second component (b) is present in the vector in the opposite orientation to the first component (a). Such vector systems have use in the treatment of diseases characherised by inflammation.

Description

GENE THERAPY

The present invention relates to a vector for use in medicine, or in the manufacture of a medicament for use in medicine.

The development of transcriptionally controlled systems which function in eukaryotic cells are important for achieving regulated gene expression in gene therapy. The tetracycline system for regulated gene expression was originally developed as a two vector 'off' system in which a chimeiic tetracycline transactivator (tTA), composed of tetR and NP16 is constitutively expressed from one vector, and binds to a tetracycline operon (tetO) containing promoter (Ptet) in a second vector inducing gene expression (Gossen & Bujard, Proc. Natl. Acad. Sci. USA 89 5547-5551 (1992)).

When tetracycline is added it binds tTA and induces a conformational change which prevents it from binding to the tetO and thus switches off gene expression. The 'off system displays regulated expression in excess of 1000-fold when the two vectors are stably transfected into HeLa cells. Several studies have since reported adaptations of the original tetracycline 'off system which facilitate application in gene therapy settings.

One variation involves combining the components of the two vectors in a single self- contained vector, which avoids the need to deliver two vectors to a single cell, this combination has been reported with both retroviral (Paulus et al, J. Virol. 70 62-67 (1996); Lindermann et al, Mol. Med. 3 466-476 (1997)) and plasmid vectors (A-Mohammadi et al, Gene Therapy 5 76-84 (1998)). Another modification has been expressing tTA under the control of a tetO promoter (Ptet). In this autoregulatory format the tTA regulates its own expression, in the absence of tetracycline the tTA is able to interact with the tetO promoter (Ptet) and can therefore upregulate its own expression in a positive feedback loop. When tetracycline is added to the system the tTA can no longer interact with the promoter and so tTA expression is down regulated. The advantage of autoregulated expression of tTA is low level expression of tTA in the presence of tetracycline, which should reduce toxic effects of constitutively expressed tTA, and higher levels of inducible gene expression when tetracycline is removed.

The autoregulated expression of tTA was first adopted in a two vector system (Shocked: et al, Proc Natl Acad Sci USA 92: 6522-6526 (1995)) and was later incorporated into self-contained vectors (A-Mohammadi & Hawkins Gene Therapy 5: 76-84 (1998); Hofmann et al, Proc Natl Acad Sci; 93: 5185-5190 (1996)). In the 'off system tTA autoregulation results in high level expression of tTA in the absence of antibiotic. However, it has been postulated that tTA may have a 'squelching' effect at high intracellular levels (Gossen & Bujard, Proc Natl Acad Sci USA; 89: 5547-

5551 (1992)). Indeed this view was supported by a study which indicates that continued high level tTA expression from an autoregulatory vector is detrimental to cell functions including growth and the cell cycle (Gallia & Khalili, Oncogene ; 16: 1879-1884 (1998)). By contrast cells maintained in the presence of tetracycline displayed normal growth characteristics.

The toxic effects of tTA are usually attributed to potential 'squelching' effect of the NP16 component, but the tetR component could also contribute to these effects. A study examining tetR expression in tomato plants, demonstrated that high levels of the TniO encoded tetR above a threshold level which displayed toxic effects including reduced leaf chlorophyll content, leaf size and root dry weight (Corlett et al, Plant, Cell and Environment ; 19: 447-454 (1996)). Interestingly, supplementing the sand with tetracycline significantly reduced the deleterious effects in plants.

Autoregulated expression of tTA in self-contained vectors requires tetracycline (Tc) or tetracycline derivatives to switch off the tTA-dependant expression unit. Even though Tc and many Tc derivatives are non-toxic to eukaryotic cells at the low concentrations required to abolish gene expression, their continuous presence is undesirable in a variety of experimental set-ups, for example, in the breeding of transgenic animals and in gene therapy. Moreover, the induction of gene expression may be slower when an effector needs to be removed, a feature which is disadvantageous in situations where the kinetics of switching on/off gene expression play a role, for example, in developmental processes.

Gossen and co-workers later developed a two-vector tetracycline 'on' system. Mutation of the tTA produced the reverse tetracycline transactivator (rtTA) which induces gene expression in the presence of doxycycline (Gossen et al, Science 268 1766-1769 (1995)).

An autoregulatory expression vector that utilises the Ptet promoter and rtTA transactivator has also been described in WO 01/59088.

However, despite progress in the field, there remains a need for an improved gene therapy vector that is both safe and effective. Regulated expression from the "on" system appears to be compromised due to the basal activity of the Ptet element and inherent binding of the rtTA to Ptet. The present invention offers a solution to this problem in a vector that is under the control of a regulated promoter system and which delivers reliable expression of the therapeutic protein or proteins of interest.

According to a first aspect of the invention, there is provided a vector comprising: a first component (a) comprising a first promoter element composed of a tetracycline-regulated promoter upstream of a nucleic acid sequence encoding a first protein in which said nucleic acid sequence is upstream of a termination of transcription signal; and a second component (b) comprising a second promoter element upstream of a nucleic acid sequence encoding the reverse tetracycline transactivator (rtTA) protein rtTA2S-M2 which is upstream of an internal ribosomal entry site (IRES) which is upstream of a nucleic acid sequence which encodes the tetracycline repressor protein tetR-KRAB, which is upstream of a termination of transcription signal in which the second component (b) is present in the vector in the opposite orientation to the first component (a).

Regulated expression from the "on" system previously used is compromised due the basal activity of the Ptet element and inherent binding of the rtTA to the Ptet. These deficiencies have been surmounted in the present invention with the use' of an improved transactivator and a tetR targeted repressor. The improved transactivator rtTA2S-M2 shows increased cellular stability and enhanced tetO binding characteristics compared with rtTA. The repressor is a fusion of tetR and Kruppel- associated box (KRAB), a zinc finger repressor domain derived from Kox-1 and present at the NH₂ terminal of several proteins. KRAB functions by recruiting a co- repressor KAP-1 which interacts with proteins such as HP1 (non-histone heterochromatin-associated protein) and this leads to the formation of a heterochromatin-like complex. In the absence of doxycycline (Dox), tetR-KRAB binds to the Ptet and effectively prevents all transcription from the promoter.

An IRES is an internal ribosome entry site. They have been identified in mRNA from viruses and eukaryotic cells. Viral examples include encephalomyocarditis virus (EMCN), foot-and-mouth disease virus (FMDN), poliovirus, hepatitis C virus and a mammalian example is vascular endothelial growth actor (NEGF). Whilst there is not a common sequence to these different IRES's, their secondary structure enables the function of the IRES to be exhibited.

Suitably, a termination of transcription signal may be provided upstream of the first promoter element. In some embodiments, it may be preferred that the second promoter is a constitutive promoter.

In another preferred embodiment, the first promoter element may be bi-directional. In such an arrangement, it may be convenient for the bi-directional first promoter to be upstream of a nucleic acid sequence encoding a second protein which is in the opposite orientation to the nucleic acid sequence encoding the first protein. In some circumstances, the vector of this aspect of the invention may be composed of: a first component (a) in which the first promoter element is the Ptet promoter upstream of a nucleic acid sequence encoding a first protein in which said nucleic acid sequence is upstream of a termination of transcription signal; and a second component (b) in which the second promoter element is a constitutive promoter upstream of a nucleic acid sequence encoding the reverse tetracycline transactivator (rtTA) protein rtTA2S-M2 which is upstream of an internal ribosomal entry site (IRES) which is upstream of a nucleic acid sequence encoding a tetracycline repressor protein, preferably tetR-KRAB, which is upstream of a termination of transcription signal.

Suitably, a termination of transcription signal may be provided upstream of the first promoter element. As described above, the first promoter element may be bidirectional and in such arrangements the bi-directional first promoter may also be upstream of a nucleic acid sequence encoding a second protein which can be in the opposite orientation to the nucleic acid sequence encoding the first protein.

The term "vector" generally refers to any nucleic acid vector which may be RNA, DNA or cDNA. The vector can be described alternatively as an "expression vector".

The terms "vector" or "expression vector" may include, among others, chromosomal, episomal, and virus-derived vectors, for example, vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SN40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. Generally, any vector suitable to maintain, propagate or express nucleic acid to express a polypeptide in a host may be used for expression in this regard. The vector may be constructed from a bacterial plasmid, for example the bacterial plasmid pUC18.

Preferred arrangements of vectors according to the present invention are vector pGTMIKTZ as shown in Figure 6 and vector pGTMLKT as shown in Figure 7. An example of a vector comprising a bi-directional promoter as described above is shown in Figure 8.

In certain embodiments of the invention, the vectors may provide for specific expression. Such specific expression may be inducible expression or expression only in certain types of cells or both inducible and cell-specific. Preferred among inducible vectors are vectors that can be induced for expression by environmental factors that are easy to manipulate, such as temperature, nutrient additives, hypoxia and/or the presence of cytokines or other biologically active factors. Particularly preferred among inducible vectors are vectors that can be induced for expression by changes in the levels of chemicals, for example, chemical additives such as antibiotics. A variety of vectors suitable for use in the invention, including constitutive and inducible expression vectors for use in prokaryotic and eukaryotic hosts, are well known and employed routinely by those skilled in the art.

Recombinant expression vectors will include, for example, origins of replication, a promoter preferably derived from a highly expressed gene to direct transcription of a structural sequence, and a selectable marker to permit isolation of vector containing cells after exposure to the vector.

Mammalian expression vectors may comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation regions, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking non-transcribed sequences that are necessary for expression. Preferred mammalian expression vectors according to the present invention may be devoid of enhancer elements. The promoter sequence may be any suitable known promoter, for example the human cytomegalovirus (CMV) promoter, the CMN immediate early promoter, the HSN thymidine kinase promoter, the early and late SN40 promoters or the promoters of retroviral LTR's, such as those of the Rous sarcoma virus ("RSN"), and metallothionein promoters, such as the mouse metallothionein-I promoter. The promoter may comprise the minimum sequence required for promoter activity (such as a TATA box without enhancer elements), for example, the minimal sequence of the CMN promoter (mCMV). Preferably the promoter is a mammalian promoter that can function at a low basal level devoid of an enhancer element.

Preferably, the promoter is contiguous to the first and/or second nucleic acid sequence. In practice, the promoter lies between the tet operator sequence and the first or second nucleic acid sequence.

The tet operator sequence of the expression vector of the first aspect of the invention may comprise seven tet operators located upstream from the sequence of the promoter. It is contemplated that variants, for example, homologues or orthologues, of the promoters described herein are part of the present invention.

In some situations it may be desirable for the tet operator sequence to be devoid of binding sites for transcription factors which effect the level of basal activity in the expression system. In such situations, the tet operator may be devoid of nucleic acid sequences which bind to the GAT A sequence of transcription factors. Moreover, the tet operator which is devoid of GATA binding sites may retain a suitable recognition sequence for the tet repressor (tetR). In this context the transcription factors may be endogenous transcription factors.

Preferably, the backbone of the expression vector of the first aspect of the invention is derived from a vector devoid of its own promoter and enhancer elements, for example the plasmid vector pGL2. Enhancers are able to bind to promoter regions situated several thousands of bases away through DΝA folding (Rippe et al TIBS 1995; 20: 500-506 (1995)). In the event of an interaction between enhancer elements of promoters in self contained vectors, rtTA may be prevented from binding to the tetO elements of the Ptet, or the expression of rtTA from the adjacent promoter, for example CMN promoter, may in some way be reduced, or its basal level increased.

The expression vectors may also include selectable markers, such as antibiotic resistance, which enable the vectors to be propagated.

In the present application, the promoter sequence and tet-operator sequence are referred to as "Ptet".

The Ptet sequence may be adjacent to the nucleic acid sequences encoding the first protein or the first and second protein. Preferably, the Ptet sequence is suitably operably linked to the nucleic acid sequences encoding the first protein or first and second protein. In practice, the Ptet sequence controls the expression of the nucleic acid sequence of the first protein or the first and second protein. In this situation, in the presence of an effector, rtTA binds to the tet operator sequence inducing expression of the first and second nucleic acid sequence.

The nucleic acid sequences of the vector of the invention may encode a reporter protein as described above, such as a chloramphenicol acetyl transferase ("CAT") transcription unit, luciferase or green fluorescent protein (GFP). The application of reporter genes relates to the phenotype of these genes which can be assayed in a transformed organism and which is used, for example, to analyse the induction and/or repression of gene expression. Reporter genes for use in studies of gene regulation include other well known reporter genes including the lux gene encoding luciferase which can be assayed by a bioluminescence assay, the uidA gene encoding β- glucuronidase which can be assayed by a histochemical test, the lacZ gene encoding β-galactosidase which can be assayed by a histochemical test, the enhanced green fluorescent protein which can be detected by TJN light, UN microscopy or by FACS.

The DΝA comprising the nucleic acid sequence of the invention may be single or double stranded. Single stranded DΝA may be the coding or sense strand, or it may be the non-coding or anti-sense strand. For therapeutic use, the nucleic acid sequences are in a form capable of being expressed in the subject to be treated.

The termination sequences in the vector may be a sequence of adenylate nucleotides which encode a polyadenylation signal. Typically, the polyadenylation signal is recognisable in the subject to be treated, such as, for example, the corresponding sequences from viruses such as, for human treatment, the SN40 virus. Other termination signals are well known in the art and may be used.

Preferably, the polyadenylation signal is a bidirectional terminator of RΝA transcription. The termination signal may be the polyadenylation signal of the simian 40 virus (SN40), for example the SN40 late poly(A). Alternatively, the termination sequence may be the polyadenylation signal of bovine growth hormone which results in maximal expression when combined with a CMN promoter (Yew et al Human Gene Therapy ; 8: 575-584 (1997)).

In addition the expression vector may comprise a further polyadenylation sequence, for example an SN40 early poly(A). Such a further poly (A) may be located upstream of the first nucleic acid sequence to reduce cryptic transcription which may have initiated within the vector thereby ensuring that basal gene expression from the vector is minimal.

Gene expression from integrated viral genomes may be susceptible to chromosomal positional effects. Such effects include transcriptional silencing and promoter activation by nearby heterologous enhancers. In addition, integrated sequences can activate expression of nearby genes and oncogenes. These effects are reduced through the use of elements which form boundaries to the inserted viral genome. Insulators are genetic elements such as the chicken β-globin 5' DΝase I hypersensitive site (5ΗS4) which mark a boundary between an open chromatin domain and a region of constitutively condensed chromatin. Other elements termed scaffold or matrix attachment regions (S/MAR) anchor chromatin to nuclear structures and form chromosomal loops which may have a physiological role in bringing distal regulatory elements into close proximity to a corresponding promoter. An example is located in the human mterferon-β locus and is termed the IFN-SAR. Both insulators and S MAR can reduce position effects with greatest activity demonstrated when they were combined in a lentiviral vector (Ramezani et al, Blood 101: 4717-24, (2003)). Clearly such elements can be of benefit in regulated vectors such as those described herein after they are integrated into the host genome.

The second nucleic acid sequence of the expression vector encodes reverse tetracycline transactivator (rtTA) and can be composed of the mutant Tet repressor, reverse Tet repressor (rTetR) fused to a VP16 moiety (Gossen et al, Science 268 1766-1769 (1995)). Reverse transactivator (rtTA) requires effector molecules which are tetracycline or certain tetracycline derivatives for specific DNA binding. The effector may be chlortetracycline, oxytetracycline or anhydrotetracycline or doxycycline. Preferably the effector is doxycycline (Dox). Typically, the concentration of Dox required in cells to activate rtTA binding to tet operator elements in the autoregulatory expression vector is greater than lOng/ml, preferably between lOng ml and lμg l, for example between 50ng/ml and 900ng ml, lOOng/ml and 800ng/ml, 200ng/ml and 700ng/ml, 300ng/ml and 600ng ml or 400ng/ml and 500ng/ml.

The effector molecule generally determines whether rtTA binds to the tet operator sequence. In the absence of an appropriate effector, rtTA binding to the tet operator sequence may not take place thereby preventing transcriptional activation of the first and/or second nucleic acid sequence. In contrast, in the presence of an appropriate effector, rtTA binding to the tet operator sequence may take place and transcription of the first and/or second nucleic acid sequence from the promoter sequence may occur.

Preferably, in the vector system of the present invention low level expression takes place in the absence of an effector such as Dox. Rapid "switching on" of gene expression generally takes place in the presence of the effector and gene expression is rapidly "switched off' following removal of the effector.

According to the present invention, the first or second protein encoded may be a cytokme, a chemokine, a growth factor, a differentiation factor, a peptide hormone, or a receptor for such a protein, or a derivative thereof, or an antibody, or a derivative thereof.

It may be preferred that the cytokine is an interleukin, or an interferon, or a cytokine receptor, or soluble receptor portion thereof, or a subunit thereof.

The protein encoded may include, but is not limited to, a growth factor (e.g. TGFβ, epidermal growth factor (EGLF), platelet derived growth factor (PDGF), nerve growth factor (NGF), colony stimulating factor (CSF), hepatocyte growth factor, insulin-like growth factor, placenta growth factor); differentiation factor; cytokine e.g. interleukin,

(e.g. E -1, IL-lra, IL-2, IL-3, IL-4, IL-5, T -6, E -7, IL-8, IL-9, EL-10, IL-11, EL-12, LL-12p40, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, D -19, E -20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, 1L-28, or IL-29) either or β forms thereof, interferon (e.g. IFN-α, IFN-β and IFN-γ), tumour necrosis factor (TNF), e.g. TNF-α, IFN-γ inducing factor (IGIF), vascular endothelial growth factor (VEGF), lymphotoxin α- or -β (LTα or LTβ), bone morphogenetic protein (BMP); chemokine (e.g. MIPs (Macrophage Inflammatory Proteins) e.g. MlPlα and MlPlβ; MCPs (Monocyte Chemotactic Proteins) e.g. MCP1, 2 or 3, or eotaxin; RANTES (regulated upon activation normal T-cell expressed and secreted)); trophic factors; cytokine inhibitors; cytokine receptors; free-radical scavenging enzymes e.g. superoxide dismutase or catalase; peptide mimetics; protease inhibitors; tissue inhibitor of metalloproteinase sub classes (TIMPS); angiostatin, or endostatin; and serpins (inhibitors of serine proteases). Preferably, the protein will be derived from the species to be treated e.g. human origin for the treatment of humans. Preferred cytokine receptors include the Tissue Necrosis Factor Receptor (TNF-R), in particular the soluble, extracellular domain of the TNF-R (Neve et al Cytokine, 8:365- 370 (1996)).

It may also be preferred to provide the nucleic acid encoding the first or second protein in multiple copies, thus allowing for multimeric forms of the protein to be encoded. For example, a plurality of extracellular domains of TNF-R may be encoded.

In other embodiments of the invention, it may be preferred that the first or second protein are present in the form of a fusion protein in which the biological activity of the first or second protein is rendered latent. A method for rendering biologically active proteins latent is described in WO 02/055098, in which a fusion protein is constructed from a latency-associated peptide (LAP) connected via matrix metalloproteinase (MMP) proteolytic site to a biologically active protein. The LAP adopts a conformational arrangement such that the activity of the biologically active protein is masked in vivo or in vitro. The action of a protease at the MMP cleavage site releases the biologically active protein. Preferably, the LAP is the LAP of TGF- β-1, -2, -3, -4 or -5. Suitably, the MMP cleavage site is cleaved by MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, or MMP10.

The term "latent" as used herein, may relate to a shielding effect which may hinder interaction between the fusion protein and other molecules in the cell surface. Alternatively the term latent or latency may be used to describe a reduction in the activity (up to and including ablation of activity) of a molecule/agent associated with the fusion protein. The term latency may also relate to a stabilising effect of the fusion protein. The effect may be in full or in part, i.e. partial.

In certain embodiments of the invention, the first or second protein may be a reporter protein, such as luciferase or secreted alkaline phosphatase. According to a second aspect of the invention, there is provided an expression system comprising a vector as defined above. Such a system may comprise one or more cells, which may be mammalian cells, such as non-human cells or human cells, which may derived from a cell line in culture.

Representative examples of appropriate host cells for the autoregulatory expression vector include bacterial cells, such as streptococci, staphylococci, E.coli, streptomyces and Bacillus subtilis; fungal cells, such as yeast cells, for example Saccharomyce cerevisiae, and Aspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, C127, 3T3, BHK, 293 and Bowes melanoma cells and other suitable human cells; and plant cells.

Preferably, the host cells of the expression system are mammalian cells in which a low basal activity of Ptet is observed. The basal activity of Ptet has previously been shown to be dependent upon the mammalian cell type in which it is employed, with high basal expression of genes observed in several cell lines (Freundlieb et al., J Gene Med; 1: 4-12 (1999)).

Introduction of an expression vector into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction, infection of other methods. Such methods are described in many standard laboratory manuals, such as Sambrook et al, Molecular Cloning: A Laboratory Manual, 2^nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).

Mature proteins can be expressed in host cells including mammalian cells such as CHO cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can be employed to produce such proteins using RNA's derived from the expression vector of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook et al, Molecular Cloning: A Laboratory Manual, 2^nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).

According to a third aspect of the present invention, there is provided a pharmaceutical composition comprising a vector as defined above.

According to a fourth aspect of the invention, there is provided a composition comprising a vector as claimed in any preceding claim and a pharmaceutically acceptable diluent and or adjuvant.

According to a fifth aspect of the invention, there is provided a unit dosage form comprising a vector as claimed in any preceding claim.

According to a sixth aspect of the invention, there is provided a vector as claimed in any preceding claim for use in medicine.

According to a seventh aspect of the invention, there is provided the use of a vector as claimed in any preceding claim for use in the manufacture of a medicament for use in the treatment of an inflammatory disease.

Alternatively, this aspect of the invention also extends to a method of treatment of a disease condition comprising the administration of a vector as defined in any preceding claim to a subject in need thereof.

As used herein the term "treatment" includes any regime that can benefit a human or non-human animal. The treatment may be in respect of an existing condition or disorder, or may be prophylactic (preventative treatment). The treatment may be of an inherited or acquired disease. The treatment may be of an acute or chronic condition. Preferably, the treatment is of a chronic condition with relapsing symptoms. The nucleic acid sequences of the vector that encode a protein may encode a protein for use in the treatment of a genetic disorder including, but not limited to, cystic fibrosis, cancer, haemophilia, X-linked SCID, Huntington's chorea, Addison's disease or Graves' disease. Other disorders, falling within the definition genetic disorders, are also contemplated by the present invention including, but not limited to, rheumatoid arthritis, diabetes mellitus or diabetes insipidus, multiple sclerosis, atherosclerosis, Alzheimer's disease, Parkinson's disease , Crohn's disease or any inflammatory disease.

A vector according to the first aspect, or an expression system according to the second aspect, of the invention may be used therapeutically in the method of the invention by way of gene therapy.

Administration of the vector of the invention, or the expression system of the invention, may be direct to the target site by physical methods. Examples of these include topical administration of the 'naked' nucleic acid vector in an appropriate vehicle for example in solution in a pharmaceutically acceptable excipient such as phosphate buffered saline, or administration of the vector by physical methods such as particle bombardment according to methods known in the art.

Other physical methods for administering the nucleic acid directly to the recipient include ultrasound, electrical stimulation, electroporation and microseeding. Further methods of administration include oral administration or administration through inhalation.

Particularly preferred is the microseeding mode of delivery which is a system for delivering genetic material into cells in situ in a patient. This method is described in US Patent No. 5,697,901.

Vectors according to the invention may also be administered by means of delivery vectors. These include viral delivery vectors, such as adenovirus, adeno-associated, lentivirus or retrovirus delivery vectors known in the art.

Other non-viral delivery vectors include plasmid delivery vectors, including complexes with liposome delivery vehicles known in the art. Administration may also take place via transformed host cells. Such cells include cells harvested from the subject, into which the nucleic acid is transferred by gene transfer methods known in the art, followed by growth of the transformed cells in culture and grafting to the subject.

As used herein the term "gene therapy" refers to introduction of new genetic material to the cells of an individual with resulting therapeutic benefit to the individual (somatic gene therapy). Furthermore, gene therapy can be divided into ex vivo and in vivo techniques. Ex vivo gene therapy relates to the removal of body cells from a patient, treatment of the removed cells with a vector i.e., a recombinant vector, and subsequent return of the treated cells to the patient. In vivo gene therapy relates to the direct administration of the recombinant gene vector by, for example, intravenous or intramuscular injection.

The method of gene therapy can be carried out in vivo or ex vivo, in which cells may be transformed in vitro prior to administration to an individual.

For gene therapy, the present invention may provide a method for manipulating the somatic cells of human and non-human mammals.

The present invention therefore provides a method for providing a human with a therapeutic protein comprising introducing mammalian cells into a human, the human cells having been treated in vitro to insert therein a vector, according to the present invention, encoding a therapeutic protein, the human cells expressing in vivo in the human a therapeutically effective amount of the therapeutic protein.

Each of the individual steps of the ex vivo somatic gene therapy method are also covered by the present invention. For example, the step of manipulating the cells removed from a patient with the expression vector or expression system of the present invention. As used herein, the term "manipulated cells" covers cells transfected with a recombinant vector. Also contemplated is the use of the transfected cells in the manufacture of a medicament for the treatment of a genetic disorder.

Preferred features for the second and subsequent aspects of the invention are as for the first aspect mutatis mutandis.

The invention will now be further described by way of reference to the following Examples and Figures which are provided for the purposes of illustration only and are not to be construed as being limiting on the invention. Reference is made to a number of Figures in which:

Figure 1. Expression plasmids CMV immediate-early enhancer/promoter; Luc+ improved luciferase gene; dTNFR dimeric hTNFR2; Luc luciferase gene; Ptet tetracycline responsive promoter; rtTA: reverse tetracycline transactivator; SV40 early late poly A signal (black triangle); β-globin poly A signal (grey triangle); downstream SN40 untranslated region (grey box). Numbers in parenthesis represent the length of each vector in base pairs.

Figure 2. Expression of dTNFR from pcdTNFR and pGTRTT transfected Cos-7 cells. Cos-7 cells plated in 12 well plates at 0.4x10 /well were transiently transfected with 2 μg of the constructs pGTRTT, pcdTNFR, pGTRTEmpty and pcDNA3. Cells transfected with pGTRTT or pGTRTEmpty were either cultured in normal media (white bars) for media supplemented with Dox (1 μg/ml; black bars). Levels of human TNFR2 were measured in culture supernatants collected after 48 hours and are expressed as the mean of triplicate values with vertical lines representing the SEM.

Figure 3. Constitutive and regulated luciferase expression in vivo. (A) Plasmid pcLuc+ (15 μg) was injected i.m. into the right quadriceps of 6 naϊve DBA/1 mice. The muscle of 3 mice was then electroporated (8 pulses, 200 Volts/cm, 20 ms duration, 2 Hz) whilst the other 3 mice were untreated. 3 days later the experiment was terminated and muscle processed for measurement of luciferase. Levels of luciferase are the mean of 3 animals and are normalised for protein concentration of the muscle lysate. Vertical lines represent SEM and a significant difference between p< 0.01 between the luciferase level in the muscle of electroporated and non- electroporated mice is indicated by "k. (B) Plasmid DNA (15 μg) was injected (i.m) and electroporated in naϊve DBA 1 mice. After two weeks GTRTL injected mice (n=5 for all groups) were given 10% sucrose (white bar), or Dox drinks (black bars) prepared in 10% sucrose at 200 μg/ml and 2 mg/ml for the subsequent 2 weeks. Luciferase expression in muscle was determined in dissected muscle and was compared to mice that received the control plasmids pGCMV (checked bar), pGmCMV (hatched bar) and pGTL (bricked bar). Significant differences between the pGTRTL + Dox groups and the non-induced pGTRTL group of p<0.05 are indicated by "*. (C) The down regulation of luciferase expression from pGTRTL was assessed in a group of mice that had received Dox 200 μg/ml for 2 weeks, they were switch to sucrose for 3 days after which luciferase levels in muscle were compared with groups that were non-induced or continuously induced for the duration of the experiment. Significant difference between the pGTRTL + Dox group and the non-induced pGTRTL group of p<0.05 is indicated by * and a significant difference between the pGTRTL Dox removed group and the pGTRTL Dox 200 group of p<0.05 is indicated by $.

Figure 4. Effect of pcdTNFR of on progression of CIA. Development of arthritis was monitored by clinical score (A, C and E) and hind paw swelling (B, C and F) in DBA/1 mice. Treatment was administered after onset of clinical arthritis. All mice injected with pcdTNFR (O; n=16) and pcDNA3 (D; n=ll) are depicted in A and B, whilst those with a clinical score less than 2 at the time of DNA injection are presented in C and D, and those with a clinical score above 2 when treatment was initiated are plotted in E and F. Significant differences between the pcdTNFR and pcDNA3 group of p<0.05 are indicated by " .

Figure 5. Gene therapy treatment of CIA with regulated expression of dTNFR.

Progression of CIA was monitored by clinical score (A, C and E) and hind paw swelling (B, D and F) in groups that received pGTRTEmpty + Dox (D; n=16), pGTRTT - Dox (O; n=16) and pGTRTT + Dox (•; n=17). . Treatment was administered after onset of clinical arthritis. All treated animals are illustrated in A and B, whilst those with a clinical score of 2 or less at the time of DNA injection (day 27) are illustrated in C and D, and animals with a clinical score greater than 2 at the start of treatment depicted in E and F. Significant differences between the pGTRTT + Dox and pGTRTT - Dox groups are indicated by * p<0.05, ick ρ<0.02 and *** p<0.01 respectively.

Figure 6: shows pGTMIKTZ

Figure 7: shows vector pGTMIKT

Figure 8: shows pGBTRTTL

Figures 9(a), 9(b), 9(c): showing expression of (a) dTNFR, and (b) luciferase from transfected DTFs in response to Dox (concentrations from O-lOOOng ml) induction for 48 hours. Expression of dTNFR and luciferase correlates well as shown in (c).

Examples

Materials and Methods

DNA and Cloning

The vectors pGT, pGTL, pGTE, pGTRTL, and pGCMV have been previously reported [19]. The construct pGTTRD encoding dTNFR from a Ptet was constructed by removing the dTNFR sequence from the construct pTREP [3] by restriction with Nco I filled in with Klenow, and cut with Xba I, and inserting it into pGT restricted with EcoRV and Xba I. The self-contained regulated plasmid pGTRTT encoding dTNFR was then constructed by removing the Ptet-dTNFR cassette from pGTTRD by restriction with Nhe land PflM I and ligating it into pGTRTL restricted with the same enzymes. The plasmid pcdTNFR in which dTNFR is located downstream of a CMV promoter was constructed by removing the dTNFR gene from pGTTRD by restriction with Hind III and Xba I and inserting it into pcDNA3 (Invitrogen, Leek, The Netherlands) restricted with the same enzymes.

A control construct pGTRTEmpty was also prepared which retained all the elements of pGTRTT except the dTNFR gene. pGTRTE is an autoregulatory plasmid from which EGFP expression is regulated, and was constructed by removing the Ptet-EGFP cassette from pGTE by restriction Xho I - PflMl and inserting it into pGTRTL restricted with the same enzymes. The EGFP gene was removed from pGTRTE by restriction with Cla I and re-ligation of the plasmid formed pGTRTEmpty.

The plasmid pcLuc+ which encodes luciferase from a CMV promoter was constructed by removing the improved luciferase gene (Luc+) from pGL3 Basic (Promega Corp., Madison, WI, USA) with the restriction enzymes Xho I and Xba I and inserting the gene into pcDNA3 restricted with the same enzymes.

Plasmids were expanded in E. coli DH5α, except autoregulatory vectors which were propagated in E. coli DH 21. Plasmid DNA was purified using the Plasmid Mega Eat (Qiagen Ltd., Crawley, West Sussex, UK) or when required for injection into mice the EndoFree™ Plasmid Mega Kit (Qiagen Ltd) was used. All plasmids that were used in expression studies are depicted schematically in Figure 1.

Cells.

Cos-7 (SV40 transformed monkey kidney fibroblast, ECACC Cat.No. 87021302) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (BioWhittaker, Wokingham, UK) supplemented with 10% FCS (GibcoBRL, Paisley, UK), glutamine (2 mM) (BioWhittaker), penicillin (100 U/ml) (BioWhittaker) and streptomycin (100 μg/ml) (BioWhittaker).

Transfections Transfections were performed using the calcium phosphate precipitation method used previously [19]. Cos-7 cells were plated on 12 well plates at a density of 0.4xl0⁶ per well and were transfected the next day with 2 μg of DNA. Cells were subjected to an osmotic shock on the second day, after which fresh media was added with or without Dox at a concentration of 1 μg/ml. Supernatants were collected 24 or 48 hours later and levels of dTNFR were determined by ELIS A.

In Vivo Electroporation

Mice were treated according to approved Home Office and institutional guidelines. Naϊve or arthritic DBA/1 mice were injected i.p. with the muscle relaxant Hypnorm™ (Janssen Animal Health, Jannsen Pharmaceuticals, Belgium) and were anaesthetised with Halothane (Concord Pharmaceuticals Ltd, Essex, UK) using Boyle's Apparatus (British Oxygen Company, London, UK). The fur covering the right quadracep was shaved and the exposed skin sprayed with disinfectant. Endotoxin free plasmid for injection was prepared in a solution of 0.9% NaCl at a concentration of 250 μg/ml for reporter gene studies or 833 μg/ml for therapeutic studies. DNA (20 μl) was injected i.m. at three sites, Camcare ECG gel (Camcare Gels, Mepal, Cambridgeshire, UK) was then applied to the surface of the skin. Caliper electrodes 384L (BTX Instrument Division Harvard Apparatus Inc. Holliston, MA) were applied transversely across the quadriceps and the muscle was electroporated with 4 pulses at 200 Volts/cm and 20 ms duration at a frequency of 2 Hz using a BTX Electro Square Porator ECM 830 (Harvard Apparatus Inc.), the polarity of the electrodes was then reversed and the procedure repeated.

Regulated Expression of Luciferase In Vivo

Regulated expression of luciferase from pGTRTL was assessed in DBA/1 mice following i.m. injection and electroporation. Expression of luciferase was compared with control plasmids ρGL3 Basic, pGmCMV, pGTL and pGCMV. Plasmid DNA was injected i.m. into 10-12 week old naive DBA/1 mice along with electroporation. Luciferase expression in control groups was assessed 4 weeks after DNA injection when animals were terminated and muscle snap frozen until further processing.

The plasmid pGTRTL was injected into 30 mice which were divided into 6 groups of 5. These mice received normal drinking water for 2 weeks, then 2 groups were given distilled water containing 10% sucrose, 3 groups drank Dox (200 μg/ml) and 1 group were given Dox (2 mg/ml) these Dox solutions were prepared in distilled water containing 10% sucrose. All drinking bottles were wrapped in aluminium foil and were renewed every 2-3 days. After a further 2 weeks a group of animals from each treatment group was terminated and the quadriceps muscles dissected and snap frozen. In order to analyse the reversal of gene induction from pGTRTL following the removal of Dox a group of animals that had received Dox (200 μg/ l) were switched to the sucrose drink. Three days later the remaining mice were terminated and muscles collected as before.

Luciferase Assay

Luciferase activity in transfected muscle was determined using the luciferase assay system (Promega Corp.) Quadriceps muscles dissected from treated mice were snap frozen and stored at -70°C. Frozen muscle was processed by a method similar to that described by Hartikka et al, (1996) [20], they were firstly crushed in a freezer mill (model 6750, Spex Centriprep Inc. Metuchen, NJ. USA) and the powder was transferred to an Eppendorf tube containing 0.5 ml reporter lysis butter. The suspension was then subjected to three rounds of freeze thawing, followed by 15 minutes vortexing at 4°C. Samples were centrifuged (13,000 rpm, 5 min) and the lysate collected, pelleted material was resuspended in a second aliquot of lysis buffer and was subjected to the 15 min vortexing followed by centrifugation. The second lysate was combined with the first and luciferase activity was determined in a 20 μl aliquot of the sample which was automatically mixed with 100 μl of luciferase assay substrate and light emission measured using a MLX Microtiter® Plate Luminometer (Dynex Technologies Inc., Chantilly, Virginia, USA). Protein concentration of muscle lysate were determined using the Bradford protein assay (Bio-Rad

Laboratories Inc., Hercules, CA, USA) and values of luciferase activity were expressed as relative light units (RLU) per μg of protein.

Collagen-Induced Arthritis (CIA) DBA/1 mice aged between 10 and 12 weeks were administered Hypnorm (0.1 ml, i.p.) and were shaved at the base of the tail. Bovine collagen II (CH) was emulsified with complete Freund's adjuvant at final concentration of 2 mg/ml, and a total of 0.1 ml was injected i.d. at three sites at the base of the tail. 21 days later, a booster (0.1 ml) consisting of CH emulsified with incomplete freunds adjuvant (2 mg/ml) was injected i.d. across three sites at the tail base. A further three days later animals were injected with Lipopolysaccharide (LPS) (40 μg in 0.1 ml PBS) (E. Coli Serotype 055:B5 - Sigma-Aldrich Co. Ltd., Poole, Dorset, UK) i.p. to synchronise disease [21].

The development and progression of arthritis was monitored every 2 to 3 days and was given a clinical score based on visual signs of arthritis (0.25 - swelling in a single digit, 0.5 - swelling in more than 1 digit, 1 - swelling and erythema of the paw; 2: swelling of the paw and ankle; 3 - complete inflammation of the paw; the maximum score for each mouse was therefore 12) and the thickness of hind paws was measured using POCO 2T callipers (Kroeplin Langenmesstechnik, Schlϋchtern, Germany). Mice were monitored until 40 days after immunisation when they were terminated and blood was collected for serum, and draining lymph nodes (DLN) were collected for assessment of stimulated cytokine secretion.

Three days after injection of LPS (day 27) animals were assessed for development of arthritis. Animals with a clinical score of 0.5 or above were used in gene therapy experiments and were administered 50 μg of DNA i.m. in 60 μl at three sites and were electroporated utilising conditions described above.

Cytokine Expression from DLN cells

Inguinal draining lymph nodes were removed from mice on day 40 after immunisation. Incisions were made in the lymph nodes and cells were dispersed using nylon cell strainer (70 μm, Becton Dickinson Labware, Franklin Lakes, NJ, USA). Cell suspensions were centrifuged and resuspended at 3xl0⁶/ml in DMEM supplemented with supplemented with 10% FCS, glutamine (2 mM), penicillin (100 U/ml) and streptomycin (100 μg/ml). 6 x 10⁵ cells in 200 μl were aliquoted into wells in a 96 well microtitre plate and were stimulated with either CII (50 μg/ml) or ConA (2.5 μg/ml, Sigma-Aldrich Co. Ltd) for 48 hours after which supernatants were collected and stored at - 80°C until measurements of interferon γ (IFN γ) or E -4 were performed by ELISA (see below). Detection of dTNFR and anti-human TNFR2 by ELISA

To measure levels of dTNFR, a microtitre plate was coated with 50 J of a mouse monoclonal anti-human TNFR2 (R&D Systems, Minneapolis, MN, USA) at 4 μg/ml overnight at 4°C. Plates were washed with PBS and then blocked with 200 μl of 2% casein solution in PBS for 1 hour at room temperature. Plates were washed with PBS containing 0.05% Tween 20 (PBS/Tween) prior to incubation of standards (50 μl of human TNFR2 (R&D Systems) 1 pg/ml to 1 μg/ml) and samples (50 μl of serum or culture medium) for 3 hours at room temperature. Plates were washed extensively with PBS/Tween before incubation with 50 μl of biotinylated goat anti-human TNFR2 (R&D Systems) at a concentration of 100 ng/ml for 1 hour at room temperature. Signal was detected using the TMB microwell substrate system (Kirkegaard and Perry Laboratories Inc., Gaithersburg, MD, USA) and the reaction stopped by addition of 4M Sulphuric acid (100 μl) and absorbance measurements were performed at 450 nm using an EL 312e microplate biokinetics reader (Bio-Tek

Instruments, Inc. CA, USA). The detection limit of this ELISA was 10 pg ml.

In order to determine immunogenicity of the dTNFR an ELISA was also performed to measure anti-human TNFR2 in the sera from experimental mice. Microtitre plates were coated overnight at 4°C with 50 μl of human TNFR2 (2μg/ml). Plates were washed with PBS and then blocked with 2% casein solution in PBS for 2 hours at room temperature. Plates were then washed with PBS/Tween and then incubated with serum (diluted 1:500) or dilutions of control mouse monoclonal antibody (R&D Systems: 1 μg ml to 0.1 pg/ml) for 3 hours at room temperature. Plates were again washed with PBS/Tween and bound antibody was detected with using peroxidase conjugated sheep anti-mouse IgG (The Binding Site, Birmingham, UK), after 1 hour the signal was developed and measured as described above. The control mouse monoclonal antibody was detected down to a concentration of 0.1 pg ml

ELISA for Anti-CII IgG, Microtitre plates (96 well) were coated 50 μl of 2 μg/ml CII dissolved in TBS overnight at 4°C. After blocking for 2 hours with 2% casein (200 μl), wells were repeatedly washed with PBS/Tween and then 50 μl of 10 fold dilutions of mouse sera from 1/10 to 1/100,000,000 were applied to the wells and incubated overnight at 4°C. Total anti-collagen H IgG and IgG isotypes were quantitated using 50 μl of peroxidase conjugated sheep anti-mouse IgG, IgGl and IgG2a (The Binding Site) at a dilution of 1/5000 with PBS/Tween. Signal was detected as described above. Each plate included a standard curve of a positive serum obtained from untreated CIA mice at day 40 which was used to define arbitrary units of total IgG, IgGl and ϊgG2a anti- CH antibodies.

Measurement of IL-4 and IFNγ by ELISA

Microtitre plates were coated with 50 μl of capture antibody for IL-4 (rat anti-mouse IL-4 used at 2 μg/ml; Pharmingen, San Diego, CA, USA) or IFNγ (rat anti-mouse IFNγ used at 5 μg ml; Pharmingen), both antibodies were diluted with 0.5M carbonate/bicarbonate buffer pH 9.6, and plates were incubated overnight at 4°C. Plates were washed with PBS and then blocked with 200 μl of 2% casein solution in PBS for 1 hour at room temperature. Plates were washed with PBS/Tween prior to incubation of 50 μl of samples and standards (mIL-4, 10 ng ml to 3 pg/ml or IFNγ, 300 ng/ml to 100 pg/ml) for 3 hours at room temperature. Plates were washed extensively with PBS/Tween before incubation with 50 μl of biotinylated mouse monoclonal antibody to IL-4 (rat anti-mouse IL-4 used at 0.5 μg/ml; Pharmingen) or IFNγ (rat anti-mouse IFNγ used at 1 μg/ml; Pharmingen). Plates were again washed and then incubate with streptavidin biotinylated horseradish peroxidase complex (diluted 1:1000; Amersham). Signal was detected as described before and the limits of detection for the IL-4 and IFNγ ELIS As was 3 pg ml and 100 pg/ml respectively.

Statistical tests

Descriptive statistics and significant differences between groups were calculated using students T tests for two sample data of unequal variance (Microsoft^® Excel 98 software). Results

Expression of dTNFR in vitro Transient transfection of Cos-7 cells was used to demonstrate expression of dTNFR from the construct pcdTNFR (Figure 2). A basal level of TNFR2 was also detected in the control transfection of Cos-7 with pcDNA3 which indicates that the ELISA cross reacts with the monkey TNFR2 produced spontaneously by this cell line. It has been found previously that this ELISA reacts with monkey TNFR2 (unpublished observation).

Expression of dTNFR from pGTRTT was determined in transiently transfected Cos-7 cells. Induction of transfected Cos-7 cells with Dox (1 μg/ml) for 48 hours achieved expression levels of dTNFR from pGTRTT which were equivalent to the levels produced from cells transfected with pcdTNFR (Figure 2). As previously demonstrated for pGTRTL, there was some basal activity of the Ptet in pGTRTT with dTNFR expression detected in the absence of Dox induction. As expected, the control vector pGTRTEmpty expressed no dTNFR above background in either the absence or presence of Dox.

Expression of luciferase following in vivo plasmid injection and electroporation. Electroporation has been reported to enhance the transfection efficiency of plasmid DNA delivered in vivo by i.m. injection [10]. h order confirm this effect in naϊve DBA/1 mice the constitutively expressing plasmid pcLuc+ was injected i.m. and electroporated. Measurement of luciferase expression 3 days later indicated that electroporation enhanced transfection efficiency, with expression levels 245 fold higher when compared to DNA injection alone (Figure 3a).

The self-contained autoregulatory plasmid pGTRTL, previously shown to function efficiently in vitro, was assessed for function in vivo following delivery to naϊve

DBA/1 mice. Expression of luciferase from pGTRTL was compared with luciferase expression from the control vectors pGCMV, pGmCMV, and pGTL. In each case, after DNA was injected and electroporated the mice were left for 2 weeks and then those that were injected with pGTRTL were either maintained non-induced with a drink of 10% sucrose, or were induced with Dox drinks prepared in 10% sucrose at concentrations of 200 μg/ml or 2 mg/ml. After a further 2 weeks mice were terminated and expression levels of luciferase determined. Results in Figure 3a confirm that regulated expression of luciferase from pGTRTL is observed in vivo with a 43 fold and 27 fold induction observed in groups that received Dox drinks of 200 μg/ml and 2 mg/ml respectively. Induced levels of luciferase from pGTRTL with 200 μg/ml Dox exceeded the expression levels observed with pGCMV by more than 3 fold. Interestingly, basal expression of luciferase from pGTRTL was significantly higher than from the control vectors pGmCMV, and pGTL.

The down regulation of luciferase expression from pGTRTL in vivo was assessed in mice that were untreated for 2 weeks after DNA injection, then induced with Dox (200 μg/ml) for 2 weeks and then removed from Dox for 3 days before terminating the experiment. Figure 3c illustrates that removal of Dox for 3 days resulted in return to the basal levels of luciferase expression observed for the non-induced pGTRTL group, whilst a group that continued to receive Dox for the extra 3 days maintained induced levels of luciferase expression.

Treatment of CIA with pcdTNFR after disease onset.

Control plasmid pcDNA3 (n=ll) or the expression plasmid pcdTNFR (n=14) were delivered by i.m. injection and electroporation to arthritic (clinical score at least 0.5) DBA/1 mice on day 27 after CH immunisation. Clinical score and hind paw swelling was monitored and is shown in Figure 4a and b. The results indicated that there was no therapeutic effect of pcTNFR treatment. The clinical score of mice at the time of DNA delivery on day 27 was in the range 0.5 to 4, and a published study indicates that the therapeutic outcome of TNFα inhibition is related to the level of disease activity when treatment is initiated in mouse CIA [22]. Animals were therefore sub- divided into those that had a clinical score of 2 or less, and those with a higher clinical score at the time of DNA injection, h terms of clinical score significant therapeutic effect of pcdTNFR was observed in mice (n=6) with lower disease activity at the time of DNA injection compared to those injected with pcDNA3 (n=9), and no therapeutic effect was observed in mice with a clinical score greater than 2 when treatment (pcdTNFR n=8, pcDNA3 n=2) was initiated (Figure 4c and 4e). Data for paw swelling also illustrates significant therapeutic effect of pcdTNFR in mice with lower disease activity compared with those that had more established disease at the initiation of treatment (Figure 4d and 4f).

Inhibition of CIA with dTNFR expressed from pGTRTT delivered after disease onset

Animals injected with the plasmid pGTRTT on day 27 and administered Dox (n=17) developed significantly reduced arthritis measured by paw thickness and clinical score when compared with control animals that received pGTRTT but no Dox (n=16) and animals that received the control vector pGTRTEmpty and Dox (n=16) (Figure 5 a and 5b). When therapeutic effect was assessed on the basis of disease activity at the time of DNA delivery it was clear that animals with a clinical score less than 2 responded to pGTRTT + Dox treatment both in terms of clinical score and hind paw swelling (Figure 5c and d). Where disease had progressed to give a clinical score greater than 2 at the time of DNA delivery no benefit of pGTRTT + Dox was observed (Figure 5e and ).

Levels of dLTNFR in the serum of pGTRTT treated mice was below the detection level of the ELISA (data not shown), and no anti-human TNFR2 was detected in these sera (data not shown) indicating that the dTNFR was not immunogenic during the time frame of the experiment.

The average daily fluid intake per mouse for the groups in which the treated animals were housed were determined at 3.6 ml, 3.1 ml and 3.4 ml for the treatment groups pGTRTT + Dox, pGTRTT - Dox and pGTRTEmpty + Dox respectively. The average amount of Dox administered was therefore 720 μg in the pGTRTT treated group, which equates to approximately 30 mg kg Dox/day. Immunological status of CIA mice was not altered by dTNFR treatment.

The anti-CII antibody profile of mice treated with pGTRTT +/- Dox and pGTRTEmpty was similar at the end of the experiment for all groups Table I. In addition cytokine release from DLN cells in response to CH or Con A stimulation was also unaffected by any treatment Table I.

Table I Anti-CII antibodies and cytokine release from DLN cells of CIA mice.

Treatment Anti-Collagen Antibodies Total IgGl IgGl IgG2a Ratio IgG2a/IgGl GTRTEmpty +Dox 0.64±0.11 2.0±0.7 1.6±0.3 1.3±0.4 GTRTT - Dox 1.1±0.4 1.7±0.5 1.5±0.5 1.4±0.3 GTRTT + Dox l.l±Q.3 1.6±0.3 1.3±0.2 1.4±0.4

Treatment Cytokine Release From Draining Lymph Node Cells IFNγ (ng/ml) IFNγ (ng/ml) IL-4 (pg/ml) IL-4 (pg ml) 48 hr Cπ 48 hr ConA 48 hr Cπ 48 hr ConA

GTRTEmpty +Dox 0.41±0.03 4.2±1.1 11.9±3.2 33.2+8.8

GTRTT - Dox 0.66±0.15 2.4±0.8 6.4±1.5 24.4±6.7

GTRTT + Dox 0.49±0.04 2.8+0.9 9.0±1.6 21.5+2.4

Results from at least 6 animals in each group

Anti-CH IgG levels in sera collected at the end of the experiment (day 40) was determined by ELISA with values expressed relative to those obtained for a pooled sample from treated mice at the same time point. Cytokine release from DLN cells was induced by 48 hours stimulation with CH or ConA and was measured by ELISA. Discussion

In this study, the therapeutic effect is demonstrated of dTNFR in CIA when expressed from plasmid DNA under the control of constitutive and regulated promoters. Importantly, the therapeutic effect was achieved when DNA was delivered after disease onset, and all the components for regulated expression were combined in a single vector.

The results confirm that in DBA/1 mice electroporation increases transfection efficiency in vivo following i.m. injection of plasmid DNA, with transgene expression detected beyond 4 weeks from pGTRTL. In agreement with other studies using mice, these observations indicate that there is long term persistence of plasmid DNA [10], and expression of the transgene luciferase [10] and rtTA [23] encoded from the autoregulatory plasmid. Regulation from pGTRTL in vivo is comparable to in vitro function [19] as demonstrated by the 43 fold increase in luciferase expression induced by 2 weeks Dox induction, and return to basal expression 3 days after removal of Dox. Interestingly, the basal expression from pGTRTL was elevated compared to pGTL in vivo, whilst the basal expression from the 2 vectors was similar in cultured fibroblasts [19], which may indicate an effect which is related to expression in skeletal muscle. Improvements in the tetracycline system have led to the development of tetR targeted repressors such as tetR-KRAB [24] and tTS [25] which have been shown to efficiently reduce the basal expression from the Ptet regulated promoters in vitro and when co-delivered to skeletal muscle [26, 27]. Incorporating the tetR-KRAB gene in the self-contained plasmid would reduce basal expression and increase the magnitude of regulation, as was recently achieved with an adenoviral vector [28].

According to data obtained in clinical trials transfection of human skeletal muscle with injected plasmid DNA does not seem to be as efficient as in mice, because only short term transgene expression has been demonstrated [29]. Enhanced transfection efficiency by electroporation has yet to be conducted in humans but observations with primates indicate beneficial effect [10]. The tetracycline gene regulatory system has also been demonstrated to function in primates, but there is evidence of immunogenicity in this species [30]. Whilst the components of the system may also be immunogenic in humans, further research will be needed to determine whether regulated expression of an immunomodulatory cytokine enables transfected cells to evade detection by the immune system.

Constitutive expression of therapeutic genes has been utilised extensively and successfully in experimental gene therapy. However, for clinical application regulated promoter systems are more flexible as they enable the level of expression to be controlled and provide a means to terminate gene expression. These features are likely requirements for gene therapy application in chronic relapsing conditions such as RA. Whilst this study demonstrates the effectiveness of the pGTRTT autoregulated vector, the full utility was not harnessed because dTNFR was continuously induced. More stringent examination requires the use of chronic arthritis models such as CIA in Vβl2 TCR tg mice [31] and use of therapeutic molecules that actually reverse established disease for example IL-4 [32] and B -1 inhibitors [22, 33].

In this study therapeutic effect of dTNFR was clearly demonstrated in CIA when expressed from plasmids with constitutive or regulated promoters, but in both cases the therapeutic effect was related to the disease severity at the time of DNA injection.

The first reports of anti-TNFα treatment by protein therapy in CIA indicated that therapeutic effect was observed when anti-TNFα treatment was initiated prior to disease onset [34] [35] or immediately after onset [35]. The therapeutic effect of anti- TNFα in CIA was later shown to relate directly to the stage of CIA development at the time treatment was initiated, with maximum effect achieved when treatment was started just after disease onset. The effect was less when administered 2 days after onset and it was ineffective when treatment was started 7 days after onset [22]. Essentially, where disease activity was lower anti-TNFα was beneficial, but when disease was more advanced TNFα inhibition was ineffective. By contrast anti-TNFα therapy causes a reversal of chronic symptoms in a large proportion of RA patients, which clearly highlights a differential outcome of anti-TNFα therapy in CIA and human disease.

The tetracycline system has previously been utilised in CIA gene therapy for regulated expression of vEL-10 [27, 36]. In one study regulated expression of vE -10 was achieved by injection of 2 AAV vectors i.m. prior to immunisation of DBA/1 mice, and Dox administration started 23 days after immunisation [36]. The study by Perez et al, 2002 involved co-injection of a single plasmid from which vIL-10 was regulated along with plasmid encoding the transcriptional silencer tTS. Dox and plasmids (i.m. and electroporated) were both delivered prior to onset of disease and resulted in a modest delay in onset and incidence of arthritis.

Dox is reported to have direct effects on inflammatory processes which could, theoretically, be of direct benefit in the treatment of arthritis. In vitro studies have shown Dox inhibits bone and cartilage breakdown [37], displays inhibition of matrix metalloproteinases (MMP's) particularly the activity of MMP-13 and MMP-8 against collagen H [38], increases iNOS mRNA degradation [39] and induces Fas/Fas L- mediated apoptosis of activated T cells [40]. However, these effects of Dox in general have an IC₅₀ in excess of 10 μg/ml whereas maximal activation of the Ptet occurs at a concentration of 1 μg/ml. Indeed, no beneficial effect of Dox treatment with the control vector pGTRTEmpty was evident in this study, and a recent clinical trial showed no benefit of Dox treatment of RA patients [41]. However, the tetracycline analogue, Minocycline administered at a similar concentration (800 μg/ml) to this study was beneficial adjuvant arthritis and CIA in rats [42].

In CIA, treatment can be started before onset of disease. Initiating treatment with anti-TNFα at this stage reduces the severity of disease that develops after antibody injection [34, 35], gene expression of dTNFR from syngeneic fibroblasts [4], TNFR1- IgG encoded from adenovirus injected intravenously [43] and anti-TNFα single chain antibody expressed locally from a CH reactive T cell hybridoma [44]. Treatment with dTNFR prior to onset of CIA also reduced the anti-CH level in sera of treated mice when expressed from syngeneic fibroblasts [4] or in a transfer model when dTNFR was expressed from arthritogenic cells used to transfer CIA to DBA/1 mice [4]. Immunological effects were also seen with soluble monomeric TNFR2 when expressed from cells transferred to SOD mice with both reduced anti-CH IgG levels [45] and changes in anti-CII subclass ratio [46] indicating a down-regulation of the

Thl response.

When gene therapy has been started at onset of disease the therapeutic effects of anti- TNFα have been less consistent. Expression of a dimeric chimeiic human TNFR1- IgG fusion protein from adenovirus was effective in a rat CIA model when delivered i.v. after onset (low level) of disease, but was ineffective when delivered intra- articularly [43]. In mouse CIA i.v. administration of adenovirus encoding the same dimeric p55 molecule at onset ameliorated disease development for 10 days, and was followed by rebound exacerbated disease [47]. Injection of retro virus encoding of TNFRl-IgG peri-articularly (p.a.) at onset of disease inhibited its progression [48].

Injection of retrovirally transduced syngeneic fibroblasts encoding dTNFR at onset of CIA did not prevent disease development [4], These previous studies have used viral vectors and with this regard the results of the present study are the first to show therapeutic effect with a TNFα inhibitor expressed from plasmid constructs.

Few studies have examined the effects of anti-TNFα on the immune system when treatment commenced at, or after onset of CIA. Expression of dTNFR from fibroblasts injected at CIA onset did not alter anti-CH levels [4]. Whilst, expression of TNFRl-Ig following injection (p.a.) of encoding retrovirus at onset in arthritic paws has been reported to reduce anti-CH levels, particularly IgG2a at 7 days after onset [48]. In this study, no effect of dTNFR treatment on anti-CH levels or cytokine expression from stimulated DLN cells has been shown, Whilst this study examines immunological markers at about the same time point as Muhkerjee et al, different TNFα inhibitors, vectors and routes of delivery were used and treatment was initiated after disease onset. The observation that expression of dTNFR for 13 days in the CIA experiments was not immunogenic is encouraging. In RA patients there has been no report of immunogenicity against Etanercept^® which also contains the extracellular domain of the human TNFR2. By contrast, expression of TNFRl-IgG encoded from adenovirus in mouse CIA led to the development of auto-antibodies to mouse TNFR1 [47].

There was some evidence that these antibodies had agonistic TNFα activity in vitro, and the immunogenicity of the TNFRl-IgG was associated with the rebound exacerbation of disease that was observed.

The overall picture that emerges is that prior to disease onset inhibition of TNFα can prevent disease onset by targeting the immune response. But after onset of disease the target for TNF inhibition is to block the cascade of inflammatory cell recruitment, for optimal effect the inhibitory molecule should be delivered as soon after disease onset as possible.

This study illustrates the potential of performing gene therapy with a single injection of regulated vector in CIA. Translating these studies to clinical application in RA patients is feasible but will demand improvements in vector function and plasmid delivery.

List of abbreviations used:, , CH = collagen π; CIA = collagen-induced arthritis; dTNFR = dimeric TNFR2; Dox = Doxycycline; DLN = draining lymph nodes; DMEM = Dulbecco's modified Eagle's medium; IFN = interferon; IL = interleukin; i.m = intra-muscular; LPS = lipopolysaccharide; MMP's = matrix metalloproteinases; p.a. = peri-articularly; RLU = relative light units; RA = rheumatoid arthritis;

TNFα = tumor necrosis factor α.

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Claims

1. A vector comprising: a first component (a) comprising a first promoter element composed of a tetracycline-regulated promoter upstream of a nucleic acid sequence encoding a first protein in which said nucleic acid sequence is upstream of a termination of transcription signal; and a second component (b) comprising a second promoter element upstream of a nucleic acid sequence encoding the reverse tetracycline transactivator (rtTA) protein rtTA2S-M2 which is upstream of an internal ribosomal entry site (IRES) which is upstream of a nucleic acid sequence which encodes the tetracycline repressor protein tetR-KRAB, which is upstream of a termination of transcription signal

in which the second component (b) is present in the vector in the opposite orientation to the first component (a).

2. A vector as claimed in claim 1, in which a termination of transcription signal is present upstream of the first promoter element.

3. A vector as claimed in claim 1 or claim 2, in which the second promoter is a constitutive promoter.

4. A vector as claimed in any preceding claim in which the first promoter element is bi-directional.

5. A vector as claimed in claim 4, in which the bi-directional first promoter is upstream of a nucleic acid sequence encoding a second protein which is in the opposite orientation to the nucleic acid sequence encoding the first protein.

6. A vector as claimed in claim 1 , comprising (a) the first promoter element is the Ptet promoter upstream of a nucleic acid sequence encoding a first protein which nucleic acid sequence is upstream of a termination of transcription signal; and

(b) the second promoter element is a constitutive promoter upstream of a nucleic acid sequence encoding the reverse tetracycline transactivator (rtTA) protein rtTA2S-M2 which is upstream of an internal ribosomal entry site (IRES) which is upstream of a nucleic acid sequence encoding a tetracycline repressor protein, preferably tetR-KRAB, which is upstream of a termination of transcription signal.

7. A vector as claimed in claim 6, in which a termination of transcription signal is also upstream of the first promoter element,

8. A vector as claimed in claim 6 or claim 7, in which the first promoter element is bi-directional.

9. A vector element as claimed in claim 8, in which the bi-directional first promoter is upstream of a nucleic acid sequence encoding a second protein which is in the opposite orientation to the nucleic acid sequence encoding the first protein.

10. A vector as claimed in any preceding claim, in which the first or second protein encoded is a cytokine, a chemokine, a growth factor, a differentiation factor, a peptide hormone, or a receptor for such a protein, or a derivative thereof, or an antibody, or a derivative thereof.

11. A vector as claimed in claim 10, in which the cytokine is an interleukin.

12. A vector as claimed in claim 11, in which the cytokine is an interferon.

13. A vector as claimed in claim 10, in which the receptor is a cytokine receptor.

14. A vector as claimed in claim 10 in which the cytokine receptor is TNF-R.

15. A vector as claimed in claim 14, in which TNF-R comprises the extracellular soluble domain of TNF-R.

16. A vector as claimed in claim 15, in which a plurality of extracellular soluble domains of TNF-R are encoded.

17. A vector as claimed in any of claims 5 to 16, in which the first or second protein is present in the form of a fusion protein in which the biological activity of the first or second protein is rendered latent.

18. A vector as claimed in any preceding claim, in which the first or second protein is a reporter protein.

19. A vector as claimed in claim 18, in which the reporter protein is selected from the group consisting of luciferase and secreted alkaline phosphatase.

20. An expression system comprising a vector as defined in any one of claims 1 to 19.

21. An expression system as claimed in claim 20 which comprises one or more cells.

22. An expression system as claimed in claim 21, in which the cells are mammalian cells.

23. A pharmaceutical composition comprising a vector as claimed in any one of claims 1 to 19.

24. A composition comprising a vector as claimed in any one of claims 1 to 19 and a pharmaceutically acceptable diluent and/or adjuvant.

25. A unit dosage form comprising a vector as claimed in any one of claims 1 to 19.

26. A vector as claimed in any one of claims 1 to 19 for use in medicine.

27. The use of a vector as claimed in any one of claims 1 to 19 for use in the manufacture of a medicament for use in the treatment of an inflammatory disease.

28. A method of treatment of a disease condition comprising the administration of a vector as defined in any one of claims 1 to 19 to a subject in need thereof.