WO1999045126A2 - Activation amelioree de promedicaments - Google Patents

Activation amelioree de promedicaments Download PDF

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Publication number
WO1999045126A2
WO1999045126A2 PCT/GB1999/000672 GB9900672W WO9945126A2 WO 1999045126 A2 WO1999045126 A2 WO 1999045126A2 GB 9900672 W GB9900672 W GB 9900672W WO 9945126 A2 WO9945126 A2 WO 9945126A2
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Prior art keywords
vector
prodrug
cells
cell
nucleic acid
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WO1999045126A3 (fr
Inventor
Ian James Stratford
Adam Vorn Patterson
Susan Mary Kingsman
On Kan
Leigh Griffiths
Kyriacos Mitrophanous
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Oxford Biomedica UK Ltd
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Oxford Biomedica UK Ltd
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Priority claimed from GBGB9804841.6A external-priority patent/GB9804841D0/en
Priority claimed from GBGB9818103.5A external-priority patent/GB9818103D0/en
Priority claimed from GBGB9902081.0A external-priority patent/GB9902081D0/en
Application filed by Oxford Biomedica UK Ltd filed Critical Oxford Biomedica UK Ltd
Priority to AU32668/99A priority Critical patent/AU3266899A/en
Priority to JP2000534657A priority patent/JP2002505341A/ja
Publication of WO1999045126A2 publication Critical patent/WO1999045126A2/fr
Publication of WO1999045126A3 publication Critical patent/WO1999045126A3/fr
Anticipated expiration legal-status Critical
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/44—Oxidoreductases (1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6891—Pre-targeting systems involving an antibody for targeting specific cells
    • A61K47/6899—Antibody-Directed Enzyme Prodrug Therapy [ADEPT]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0077—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with a reduced iron-sulfur protein as one donor (1.14.15)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/14—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with reduced flavin or flavoprotein as one donor, and incorporation of one atom of oxygen (1.14.14)
    • C12Y114/14001—Unspecific monooxygenase (1.14.14.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/027—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a retrovirus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to an agent for activating prodrugs in target cells.
  • the present invention also relates to methods for activating prodrugs using the novel agents.
  • Enzyme prodrug therapy relates to the use of enzymes to activate prodrugs in therapy.
  • Prodrugs are usually pharmacologically inert or relatively inert compounds, which can be converted in vivo to active species having a therapeutic effect (reviewed by Connors 1995 Gene Therapy 2, 702; Springer and Niculescu-Duvaz 1996 Adv Drug Deliv Res 22, 351).
  • One particular use of EPT is in the treatment of tumours.
  • prodrugs were developed that exploit the body's innate enzymes to achieve activation, for example Cyclophosphamide (CP) and its isomer, Ifosphamide (IF) are activated by the P450 family of enzymes to produce nitrogen mustards that damage DNA; Mitomycin C (MMC) is activated primarily by NADPHxytochrome c (P450) reductase to produce a potent alkylating agent (e.g. Bligh et al 1990, Cancer Research 50, 7789).
  • the therapeutic index of these first prodrugs depends not upon selective delivery to the target cell but upon the differential susceptibility of the target cells to DNA damage.
  • tumours have not been shown to possess particularly high levels of enzymes that can activate this class of prodrugs (Forrester et al 1990 Carcinogenesis 11, 2163). It is thought that prodrugs such as CP, IF and MMC are activated to the therapeutic metabolite predominantly in the liver and the therapeutic metabolite is then distributed to tumour sites via the circulation. Even when tumours possess some capacity to metabolise prodrugs it is thought that down regulation of the activity contributes to drug resistance (e.g. Bligh et al 1990 ibid.).
  • tumour cells are relatively inefficient at metabolising prodrugs
  • the concept of delivering normal human enzymes directly to the tumours has lead to the concept of delivering normal human enzymes directly to the tumours in order to achieve a high local concentration of the activated compound (e.g. Chen et al 1996 Cancer Research 56, 1331).
  • transferring the genes that encode P450 directly into the tumour cells using viral vectors can enhance the efficacy of CP such that the tumour shows marked growth reduction and regression (Chen et al 1996 ibid.).
  • the relevant enzyme activities can be selectively targeted to tumour cells by one of several means. In one approach a tumour selective monoclonal antibody or fragment such as a single chain antibody
  • scFv is conjugated to the enzyme either post-synthetically or by producing a recombinant protein.
  • the protein is administered to the patient and localises at the tumour by virtue of specific recognition of the tumour surface by the antibody.
  • the non-toxic prodrug is adminstered and can only be activated at the site of the tumour by the bound conjugate (reviewed by Bagshawe 1987 Br J Cancer 56, 531). This approach is referred to as ADEPT (antibody dependent prodrug activation).
  • ADEPT antibody dependent prodrug activation
  • Another approach is to deliver the DNA encoding the enzyme to the target cell using either DNA based delivery systems such as plasmids (gene dependent enzyme prodrug therapy GDEPT) (e.g.
  • tumour specificity may be conferred by incorporating targeting ligands into the delivery vehicle or by using tumour specific promotors (e.g. Harris et al 1994 Gene Therapy 1, 170 and refs therein).
  • tumour specific promotors e.g. Harris et al 1994 Gene Therapy 1, 170 and refs therein.
  • a recent development describes a combination of the ADEPT and GDEPT approaches whereby a gene fusion is constructed such that the gene encoding the antibody-targeting moiety is fused to the gene encoding the prodrug activating enzyme.
  • the gene fusion is delivered to the target tissue either as DNA or in a gene delivery vehicle such as a viral vector (WO98/55607).
  • Another class of prodrugs shows a selective toxicity to tumours by virtue of the reductive activation that occurs in the severe hypoxic environment that is a unique physiological feature of tumours.
  • Hypoxia is the condition of abnormally low levels of oxygen and is found in most solid tumours beyond about 1mm in diameter (Coleman 1988 J Nat Cane Inst 80, 310; Vaupel et al Cancer Res 49, 6449).
  • the hypoxic environment is conductive to reductive events that can generate reduced derivatives of a variety of chemical groups (Workman and Stratford 1993 Cancer and Metastasis Reviews, 12, 73-82).
  • the prototypical bioreductive drugs are the antibiotics,
  • MMC Mitomycin C
  • POR Porfiromycin
  • N-oxides such as Tirapazamine (TRZ) (Zeeman et al 1986 Inst J
  • Tirapazamine (TRZ; 3-amino-l, 2,4-benzotriazine-l, 4-dioxide). It is metabolised to a mono-N-oxide (SR4317) and to a lesser extent to the free base SR4330. Neither the two or four electron reduction products are genotoxic and therefore one electron radical of Tirapazamine rather than the stable metabolites is thought to be involved (A . Cahill and I.N.H. White 1990, Carcinogenisis, 11, 1407). It is thought that a nitroxide anion free radical is generated and that this acts directly upon DNA to induce DNA strand breaks (reviewed by J.M. Brown 1993 Br J Cancer 67, 1163).
  • Tirapazamine displays an enhanced cell killing in hypoxic conditions but it also displays some level of toxicity at intermediate oxygen tensions. This is valuable because the degree of hypoxia fluctuates throughout a tumour and absolutely low levels of oxygen may not be maintained for any length of time so endowing Tirapazamine with a greater tumour cell killing potential than for example Mitomycin C which is only activated at very low oxygen tensions (reviewed by J.M. Brown op cit). Tirapazamine does show toxicity at high doses due to effects on the bone marrow resulting in myelo-suppression.
  • HSVTk herpes simplex virus
  • GMV prodrug Gancyclovir
  • Gancyclovir triphosphate a nucleotide that functions to block DNA synthesis
  • ADEPT enzyme prodrug therapeutic strategy
  • the enzyme is delivered to the exterior of the cell yet in most cases the active drug must cross the cell membrane and this imposes chemical design contraints.
  • the active drug must also diffuse throughout the tumour to have an effect.
  • High level activation at one site resulting from the use of a high affinity antibody may deplete the prodrug from the bulk of the tumour and simply spill the active compound into the blood circulation thus decreasing efficacy and increasing the potential for systemic toxicity.
  • repetitive prodrug adminstration is required.
  • ADEPT strategies are flawed because they have the potential to deliver active compound into the circulation.
  • VDEPT/GDEPT strategies have an advantage if there is an intracellular cofactor because the enzyme will only activate the drug intracellularly and therefore any fortuitous release of the enzyme into the circulation will not result in systemic activation and toxicity.
  • Tk gene The mechanism is however somewhat controversial as the active metabolite, a highly charged tri-phosphate, cannot traverse cell membranes without the aid of metabolic cooperation via gap junctions (reviewed in T. Connors op. cit. and Hamel et al 1996 Cancer Res 56, 2697).
  • the Tk "bystander effect" may in fact be the result of a fortuituous and therefore unpredictable, immunological response rather than a real metabolic effect.
  • Bystander effects have been observed with cyclophosphamide activation by P450 in tumours. In this case it has been proposed that the highly toxic acreolin metabolite mediates the effect but does not cause systemic toxicity because it is relatively shortly-lived (Chen and Waxman 1995 Cancer Res 55, 581).
  • the neighbouring cells may take up an intermediate metabolite such as 4- hydroxycyclophosphamide.
  • an intermediate metabolite such as 4- hydroxycyclophosphamide.
  • a bystander effect has also been observed with the aziridin, CB 1954, which can be reduced by DT diaphorase or, with much greater efficiency by bacterial nitroreductases to produce a potent alkylating agent (Knox 1988 Biochem Pharmacol 37, 4661; Bridgewater 1997 Hum Gene Therapy 8, 709) although this has not always been observed (Clark et al 1997 Gene Therapy 4, 101-110; Patterson and Stratford, unpublished observations). Any bystander effect will be increased in proportion to the concentration of the active metabolite that is generated. It is therefore desirable to enhance the activity of the prodrug activating enzyme in order to increase the concentration of the active metabolite.
  • a prodrug activating agent comprising: a) a localisation domain; and b) a prodrug activation domain for activating a prodrug in a target cell; and wherein the localisation domain is not a tumor selective antibody.
  • the localisation domain and prodrug activation domain preferably comprise amino acid sequences, or are in the form of nucleic acid sequences encoding such domains.
  • the agent is in the form of a fusion protein.
  • protein we include peptides and polypeptides.
  • the domains may be co-administered.
  • co-adminstration we do not necessarily mean simultaneous administration. It is possible for one domain to be administered before or after the other.
  • Target cell simply refers to a cell which the agent whether native or targeted or part of a delivery system is capable of transfecting or transducing.
  • a prodrug activating agent comprising at least one expressable nucleic acid sequence coding for a cytochrome P450 wherein the or each nucleic acid sequence is operably linked to one or more constitutive expression control regulatory element(s), or one or more inducible expression control regulatory element(s).
  • the agent is in the form or a modified haematopoeitic stem cell (MHSC) which comprises the prodrug activating agent or the prodrug activation domain or has been engineered to express it.
  • MHSC modified haematopoeitic stem cell
  • a prodrug activating agent comprising a modifed haematopoeitic stem cell (MHSC) comprising at least one expressable nucleotide sequence coding for a prodrug activating domain wherein the or each nucleotide sequence is operably linked to one or more constitutive expression control regulatory elements), or one or more inducible expression control regulatory element(s).
  • the present invention also provides nucleic acid vectors comprising the nucleic acid sequence in accordance with the present invention.
  • the present invention also provides a viral vector comprising the nucleic acid sequence of the present invention.
  • the present invention also provides a prodrug activating agent, a nucleic acid vector, viral vector, or transduced cell of the present invention for use in medicine.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a prodrug activating agent, a nucleic acid vector, viral vector, or transduced cell of the present invention for use in medicine.
  • the present invention provides a method of treatment of a human or animal patient suffering from a condition such as cancer (in particular solid tumours), cerebal malaria, ischaemic heart disease or rheumatoid arthritis or of a condition characterised by ischaemia, hypoxia or low glucose.
  • a condition such as cancer (in particular solid tumours), cerebal malaria, ischaemic heart disease or rheumatoid arthritis or of a condition characterised by ischaemia, hypoxia or low glucose.
  • the present invention provides a method of producing a viral strain comprising introducing a nucleic acid sequence of the present invention into the genome of a virus.
  • the method comprises introducing the nucleic acid sequence into the genome by homologous recombination between said genome and a vector of the present invention.
  • the present invention provides a method of producing a MHSC comprising introducing a vector of the present invention into a haematopaeitic strain cell (HSC).
  • HSC haematopaeitic strain cell
  • Figure 1 A shows P450 reductase sequence
  • Figure IB shows functional domains of P450 reductase
  • Figure 2 shows derivatives of P450 reductase
  • FIG. 3 shows trans-cellular targeting sequences
  • Figure 4 shows human cytochrome P450 2B6
  • Figure 5 shows IRES
  • Figure 6 shows retroviral vectors
  • Figure 7 shows gene fusion constructs
  • Figure 8 shows a retroviral vector for conferring multiple drug sensitivity
  • FIG. 9 shows the results of macrophage delivery of P450
  • Figure 10 shows P450 PCR fragment
  • Figure 11 shows pegHRELacZ
  • Figure 12 shows pBHRELacZ
  • Figure 13 shows pBHREp450del
  • Figure 14 shows pBHREP450
  • Figure 15 shows pegHREP450;
  • Figure 16 shows pONY4;
  • Figure 17 shows pONY4.1
  • Figure 18 shows pONY4HREP450
  • Figure 19 shows pONY4.1HRE450
  • Figure 20 shows pEGASUS-4;
  • Figure 21 shows pEGASUS4P450;
  • Figure 22 shows pONY4.0P450
  • Figure 23 shows pONY4.1P450.
  • Figure 1A shows P450 reductase (P450R) sequence.
  • SEQ ID NO:l is mRNA sequence (partial, GenBank accession number: S90469); SEQ ED NO: 2 shows the amino acid sequence.
  • Figure IB shows function domains of P450 reductase.
  • Figure 2 A shows anchorless P450R.
  • SEQ ID NO: 3 shows the coding DNA sequence.
  • SEQ ED NO: 4 shows the amino acid sequence.
  • Figure 2B shows FAD and NADPH binding (FN) fragment in which SEQ ID NO: 5 shows the coding DNA sequence and SEQ ED NO: 6 the amino acid sequence.
  • Figure 3 A shows SV40 large T antigen NLS fragment.
  • SEQ ED NO: 10 shows coding DNA sequence (complement 4442-4422) and SEQ ED NO: 11 the amino acid sequence.
  • Figure 3B shows basic fibroblast growth factor (bFGF).
  • SEQ ID NO: 19 shows the coding DNA sequence for the 18kD isoform and SEQ ED NO: 20 the amino acid sequence.
  • FIG. 3C shows antennapoedia homeobox peptide (pAntp).
  • SEQ ID NO: 53 shows the coding sequence of the homeobox domain and SEQ ID NO: 54 the amino acid sequence.
  • FIG. 3D shows herpes simplex virus Type 1 (HSV-1) tegument protein VP22.
  • SEQ ID NO: 55 shows coding DNA sequence (complement sequence 106391 ... 105486 of GenBank sequence X14112) and SEQ ID NO: 56 the amino acid sequence.
  • Figure 3E shows Pseudomonas aeruginosa exotoxin A (PEA).
  • SEQ ED NO: 33 shows coding DNA sequence for domain II and SEQ ID NO: 34 the amino acid sequence.
  • FIG. 3F shows single chain Fv fragment (ScFv) for ST4 with translation initiation signal and secretory signal peptide.
  • SEQ ID NO: 25 shows DNA sequence for the translation initial signal and signal peptide of ST4-ScFv (WO98/55607) and SEQ ID NO: 27 the amino acid sequence.
  • Figure 4 shows human cytochrome P450 2B6.
  • SEQ D NO: 45 shows the mRNA sequence and SEQ ID NO: 46 the ⁇ unino acid sequence.
  • FIG. 5 shows IRES (Internal ribosome entry sequence).
  • SEQ ID NO: 52 shows IRES sequence from FMDV (R100) + Sad (at the beginning) and_Yr (at the end) linkers.
  • Figure 6 A shows PKAHRE: a MLV based single transcription unit vector. Expression of therapeutic genes is controlled by a hypoxia responsive promoter (3xPGK). The gene encoding the prodrug activating enzyme is substituted for the nlsLAcZ gene shown.
  • PKAHRE a MLV based single transcription unit vector. Expression of therapeutic genes is controlled by a hypoxia responsive promoter (3xPGK). The gene encoding the prodrug activating enzyme is substituted for the nlsLAcZ gene shown.
  • Figure 6B shows COI: an MLV based vector with the CMV enhancer replacing the MLV enhancer (shaded box).
  • the gene encoding the pro-drug activating enzyme is inserted either into the Bam/Sal/Hpa polylinker or the Stu/Xho polylinker. Alternatively different genes can be inserted into each polylinker.
  • Figure 8 shows a retroviral vector for conferring multiple drug sensitivity.
  • VP22-FN confers enhanced sensitivity to Tirapazamine.
  • P450-FN confers enhanced sensitivity to Cyclophosphamide.
  • P450-FN plus VP22-FN confers enhanced sensitivity to Mitomycin C, Tirapazamine and Cyclophosphamide.
  • the prodrug activation domain is typically a prodrug activating enzyme or an active fragment of a prodrug activating enzyme; although it may be any domain which activates a prodrug.
  • An increasing number of prodrug activating enzymes are known in the art and any of these may potentially be employed.
  • Suitable prodrug activating enzymes may be natural or engineered and include cytochrome P450, cytochrome P450 reductase, thymidine kinase, nitroreductase, cytosine deaminase, DT-diaphorase,
  • NADPH ytochrome c P450 reductase and carboxy-peptidase G 2 NADPH ytochrome c P450 reductase and carboxy-peptidase G 2 .
  • a prodrug activating enzyme may be delivered to a tumour site for the treatment of a cancer.
  • a suitable prodrug is used in the treatment of the patient in combination with the appropriate prodrug activating enzyme.
  • An appropriate prodrug is administered in conjunction with the vector.
  • prodrugs include: etoposide phosphate (with alkaline phosphatase, Senter et al 1988 Proc Natl Acad Sci 85: 4842-
  • prodrug activation enzymes for use in the invention include a thymidine phosphorylase which activates the 5-fluoro-uracil prodrugs capcetabine and furtulon; thymidine kinase from Herpes Simplex Virus which activates ganciclovir; a cytochrome P450 which activates a prodrug such as cyclophosphamide to a DNA damaging agent; and cytosine deaminase which activates 5-fluorocytosine.
  • an enzyme of human origin is used. As previously mentioned this is preferably in the form of a prodrug activating enzyme.
  • the prodrug activating enzyme used in treatment of a patient will be administered in combination with the appropriate prodrug.
  • the enzyme is a cytochrome P450 or cytochrome P450 reductase.
  • Cytochrome P450s are a superfamily of membrane-bound, heme-thiolate proteins (Nelson et al 1996 Pharmacogenetics 6: 1-42) that are present in both prokaryotes and eukaryotes. These enzymes are named as cytochromes because they form a complex with carbon monoxide under reductive conditions to yield a maximum absorbance at about 450nm. Within the cell, they are detected either in the endoplasmic reticulum (microsomes) or mitochondria. They are the key monooxygenases responsible for steroid hormones metabolism, drugs inactivation, toxins/xenobiotics detoxification and oxidation of food chemicals, environmental pollutants, carcinogens and other natural or synthetic chemicals. Moreover, some cytochrome P450s have additional isomerases, reductases, dehydrases, nitric oxide synthases or esterases activities (see Rendic & Di Carlo 1997 Drug Metab Rev 29 : 413-580).
  • cytochrome P450 Being a terminal oxidase in an electron transport chain, the enzymatic action of cytochrome P450 involves the donation of two electrons from reduced pyridine nucleotide such as NADPH. This electron transfer process requires the interaction of
  • cytochrome P450 with its redox partners (flavoproteins) NADPH-dependent cytochrome P450 oxidoreductase and cytochrome b 5 .
  • Reduced cytochrome P450 binds to oxygen and its substrate. Then this enzyme catalyses the incorporation of one oxygen atom into its substrate and the other atom of oxygen into water.
  • One typical enzymatic reaction is the incorporation of a hydroxyl group by P450 into the substrate e.g. 4-hydroxylation of cyclophosphamide by cytochrome P450 2B6.
  • the expression of cytochrome P450 protein and enzyme activity can be induced or inhibited by certain xenobiotics, organic substrates or drugs e.g. induction of CYP2B isoforms by barbiturates.
  • a definition of this enzyme includes the following:
  • cytochrome P450 as used herein includes mammalian cytochrome P450 genes such as P450 2B1, P450 2B6, P450 2A6, P450 2C6, P450 2C8, P450 2C9, P450 2C11 and P450 3A4. Each of these genes has been linked to activation of the anticancer drugs cyclophosphamide or ifosphamide.
  • the enzyme activity of cytochrome P450 can be improved by random site-directed mutagenesis or directed evolution.
  • Site-directed mutagenesis requires thorough understanding of the effects of amino acid substitution on the structure and function of cytochrome P450. In contrast, such prior knowledge of P450 is not crucial for directed evolution.
  • the frequency of beneficial mutations is generally low relative to deleterious mutations. Multiple cycles of recombination and selection may be required to combine the beneficial mutations for the improved gene.
  • Directed evolution especially DNA shuffling is therefore superior to random mutagenesis because multiple beneficial mutations rather than single beneficial mutation can be generated per round. Nevertheless, both approach requires powerful and efficient selection or screening methods to obtain superior mutants from libraries of millions of different mutants.
  • Directed evolution can be performed by random oligonucleotide mutagenesis (Horwitz & Loeb 1986 PNAS 83:7405-9), chemical mutagenesis (Sweasy & Loeb 1993 PNAS 90:4626-30), error-prone PCR (Cadwell & Joyce 1994 PCR Methods Appl. 3: SI 36-40) or DNA shuffling (Stemmer 1994a Nature 370:389-91; Zhao & Arnold 1997 PNAS 94: 7997-8000). All these methods generate libraries of DNA inserts which can be ligated into expression vector for selection or screening in bacterial or mammalian cell settings. However, techniques other than DNA shuffling are limited by the size of DNA insert that they can manipulate.
  • DNA shuffling is the appropriate method for gene improvement of P450.
  • Stemmer's method Stemmer 1994a Nature 370:389-91; Stemmer 1994b PNAS 91:10747-51
  • Related P450 coding sequences are randomly fragmented by DNase I. Fragments of 10-50bp in size are pooled and purified. Overlap extension of these fragments are carried out by repeated thermocycling without primers, in the presence of a DNA polymerase. Full length genes are then obtained by PCR in the presence of specific 5'- and 3 '-primers. In addition, point mutations are also introduced in this process by the polymerase.
  • the coding DNA sequences of related human P450 isoforms which are known to be able to metabolise oxazaphosphorines e.g. cyclophosphamide can be shuffled to obtain a better enzyme for this prodrug.
  • two or more subfamilies of mammalian P450 may be shuffled to yield an improved enzyme for cyclophosphamide or other related prodrugs.
  • rat CYP2A1 Matsunaga 1990 Biochemistry 29: 1329-41 & Genbank accession number M33312
  • mouse CYP2A4 Squires and Negishi 1988 J. Biol. Chem. 263: 4166-71 & Genbank accession number Ml 9319
  • human CYP2A6 rabbit CYP2A10 (Peng et al 1993 J. Biol. Chem.
  • the localisation domain is an intracellular localisation domain.
  • the intracellular localisation domain serves to associate the prodrug activation domain with a sub-cellular location and so increases the effectiveness of the prodrug activation domain. This in turn reduces the dose of prodrug that is required and so increases the therapeutic index.
  • the target site in the target cell is a sub-cellular site of action of the activated prodrug.
  • the fusion protein therefore delivers the prodrug activation domain to the relevant location in the target cell. This improves the effectiveness of the activated prodrug.
  • the intracellular localisation domain is typically a peptide or polypeptide which is capable of preferentially or specifically binding at a sub-cellular location, such as an organelle.
  • the intracellular localisation domain may have a specific binding partner in an intracellular membrane such as the nuclear membrane or the mitochondrial membrane.
  • the intracellular localisation domain may be specifically taken up by an organelle within the target cell, such as the nucleus or the mitochondria.
  • the intracellular localisation domain is a nuclear localisation domain.
  • Nuclear localisation domains suitable for use in accordance with the invention include nuclear localisation signals from the SV40 large T antigen, nucleoplasmin and c-myc.
  • the effectiveness of the active prodrug will be proportional to the concentration of the active metabolite at the sub-cellular site of action.
  • Many of the active compounds mediate their toxic effect via direct effects on the DNA of the tumour cell. Free radicals cause DNA strand breakage as is the case with activated Tirapazamine (TPZ) (Cahill and White op. cit.) whereas alkylating agents such as CP, Mitomycin C and CB1954 cross link DNA leading to shear upon replication (Bligh et al 1990, Cancer Res. 50, 7789; Knox et al 1988, Biochem. Pharmacol. 37, 4661).
  • TPZ activated Tirapazamine
  • the protein has an ER membrane anchor sequence and is normally found at this site (G.C.M. Smith et al 1994 Proc. Natl. Acad Sci 91, 8710).
  • the active part of the enzyme projects into the cytosol and therefore activated metabolites will be generated at the ER and should then be free to passively diffuse into the nucleus. Deletion of the ER anchor sequence does not interfere with the folding of the protein or its ability to bind co-factors and donate electrons (Smith et al op. cit.).
  • the intracellular localisation domain is a nuclear association domain.
  • a first step to enhance delivery of the metabolite to the DNA is to target the P450 reductase to the nucleus.
  • a diverse variety of peptide signals have been identified that mediate the import of proteins into the nucleus (reviewed by E.A. Nigg 1997, Nature
  • the ER anchor sequence of P450 reductase (defined as residues 1 to 60, T.D.Porter et al 1990, Biochem 29, 9814) is removed to produce an anchorless P450 reductase (alP450 reductase).
  • alP450 reductase If the anchor is replaced by a nuclear localisation signal (NLS) the P450 reductase is no longer localised to the ER but is found predominantly in the nucleus. Furthermore, the increased local concentration of P450 reductase in the nucleus increases the sensitivity of the cells to killing by Tirapazamine (TPZ).
  • NLS-alP450 reductase The derivative is referred to as NLS-alP450 reductase.
  • the P450 reductase gene is organised into functional domains that correspond to the exon structure of the gene.
  • domains that bind FMN, FAD and NADPH have been identified (T.D. Porter et al 1990 Biochem. 29, 9814). These domains are structurally and functionally discrete such that they can be expressed as protein fragments and still bind their respective cofactors. It has been shown for example that a peptide spanning residues 242 to 677 will bind FAD and NADPH and furthermore this domain (the FN fragment) can transfer electrons to a range of one- electron acceptors (Smith et al op. cit.).
  • the FN fragment contains only the active region involved in electron transfer to exogenous electron acceptors.
  • the nuclear targeting signal is fused to residues 242 to 677 of P450 reductase.
  • the resulting smaller protein has easier access to the nucleus.
  • An additional benefit is that electrons are not 'lost' by transfer to FMN and to P450 and are therefore available for transfer to exogenous electron acceptors.
  • a further benefit is that the smaller size makes room for additional genes in certain GDVs e.g. retroviral vectors where capacity is restricted.
  • the derivative is referred to as NLS-FN.
  • P450 reductase is a charged molecule and contains lysine and arginine rich regions (E.A.Shephard et al 1992, Arch. Biochem. BioPhys., 294, 168). This positive charge may favour retention in the nucleus and may even locate the protein at the DNA. However in order to optimise nuclear retention and chromatin localisation even further other fusions can be envisaged. These could for example be fusions with transcription factors, high mobility group proteins, nuclear envelope proteins or any protein with a desirable nuclear localisation.
  • the localisation domain is a transcellular localisation domain.
  • the localisation domain delivers the prodrug activation domain to neighbouring cells so increasing the number of cells that can be treated.
  • An amplification of the effects of a prodrug beyond the cell in which it is activated is called the "bystander" effect.
  • This has previously been achieved by the diffusion of the active metabolite to neighbouring cells.
  • This approach has two main disadvantages in that it will concomitantly deliver the metabolite systemically, so risking systemic toxicity and there will be a dilution of the metabolite during cell to cell transfer.
  • the present invention is particularly useful for those prodrugs where the active metabolite is poorly diffusible across membranes e.g.
  • nucleotide analogues generated by thymidine kinase and cytosine deaminase the nitrogen mustards generated by cytochrome P450 metabolism, the Tnapa_ amine radical generated by P450 reductase. This is because it provides for the activating enzyme to cross membranes rather than the metabolite.
  • Useful transcellular activation domains include the third helix domain of the Drosophila antennapoedia homeobox peptide, the Herpes simplex virus VP22 protein, basic fibroblast growth factor (bFGF/FGF2), secreted single chain antibodies (s.scFv) and the transmembrane transport signals from toxins such as the Pseudomonas aeruginosa exotoxin (PEA). Also useful are membrane translocating sequences such as Kaposi fibroblast growth factor and transcription factor kB.
  • TTS trans-cellular targeting signal
  • TTS trans-cellular targeting signals
  • TTSs are as follows:
  • bFGF Basic fibroblast growth factor
  • FGF2 Basic fibroblast growth factor
  • the bFGF receptor is up-regulated in many tumour types and interaction with bFGF results in rapid internalisation of the ligand and any macromolecule that is linked to it [Baldin et al 1990 EMBO J. 9, 1511; Sosnowski et al 1996, J. Biol. Chem. 271, 33647 and refs cited therein].
  • Fusion of bFGF to P450 reductase or the FAD/NADPH fragment produces a protein that is exported and that is taken up by any cell displaying the FGF receptor. The complex is transported to the nucleus.
  • Additional signals can be added such as a classical secretion signal and NLS to ensure efficient export form any producer cell and efficient nuclear import in any target cell.
  • the derivatives are bFGF-P450R and bFGF-FN. - pAntp. This is a 60 amino acid polypeptide corresponding to the homeobox domain contained in the third exon of the Drosophila antennapoedia protein. It has the property of penetrating cells and transferring to the nucleus (Joliot et al 1991 Proc. Nat. Acad. Sci., 88, 1864). Smaller peptides derived from pAntp also have import activity and fusion proteins can be internalised (Derossi et al 1994. J. Biol. Chem.
  • the anchorless P450 reductase is fused to a secretion signal sequence to mediate export to the homeobox domain of the Drosophila antennapoedia protein to mediate import into the nucleus of neighbouring cells.
  • the derivatives are Ant-P450 reductase and Ant-FN.
  • the VP22 protein from herpes simplex virus This is a 301 aa virion structural protein that is naturally exported from infected cells, imported by neighbouring cells, transported to the nucleus and docked onto chromatin. Proteins that are fused to VP22 are similarly exported/imported and located. [Elliott and O'Hare 1997; WO97/05265].
  • the anchorless P450 reductase is fused to VP22 and the fusion protein is distributed to the nuclei of neighbouring cells.
  • the derivatives are VP-P450 reductase and VP-FN.
  • the VP22 and P450 reductase domains are separated by a flexible linker (Somia et al see later).
  • Pseudomonas aeruginosa exotoxin A encoding the translocation domain (H) (I. Pastan and D. Fitzgerald 1991 Science, 254, 1173; J. Hwang et al 1987, Cell, 48, 129).
  • the translocation domain is fused to a targeting ligand such as a single chain antibody preferably one which recognises a tumour cell specific antigen (e.g. S.I. Chen et al 1997 Nature, 385, 78 and refs therein) and to the NLS-alP450 reductase.
  • the fusion protein is secreted, docked onto the tumour cell surface, translocated and imported into the cell.
  • the derivatives are PEA-5T4-P450 reductase and 5T4-PEA-FN.
  • the targeting ligand is a single chain antibody directed to the oncofetal antigen 5T4, but any scFv could be used for example the scFv against HER-2 (J.K. Batra et al 1992, Proc Natl Acad Sci 89, 5867).
  • the invention is not necessarily restricted to the use of single chain antibodies, it may be preferable in some cases to use a two chain antibody Fab domain.
  • MTS membrane translocating sequences
  • Another suitable trans-cellular targeting domain is the hydrophobic region of a signal peptide, usually referred to as a membrane translocating sequence (MTS).
  • MTS membrane translocating sequence
  • transcription factor kB transcription factor kB
  • Kaposi fibroblast growth factor or they can be synthetic peptides selected for an MTS function.
  • MTSs can be used as carriers to deliver short peptides into living cells (e.g. Lin et al 1995. Inhibition of nuclear translocation of transcription factor NG-kB by a synthetic peptide containing a cell membrane permeable motif and nuclear localisation sequence. J. Biol. Chem 270, 14255).
  • Such peptides can be placed at the N- or C-terminus of a peptide and have been shown to be capable of translocating full length proteins (e.g. Rojas et al 1998, Genetic engineering of fusion proteins with cell membrane permeability, Nature Biotechnology, 16, 370).
  • Kaposi fibroblast growth factor MTS was fused to a bacterial enzyme encoding glutathione-S-transferase.
  • the MTS-enzyme protein was expressed in E.coli and the purified protein was shown to be readily taken up by mouse cells.
  • the MTS is fused to all or the active part of prodrug activating enzyme such as P450 reductase or nitroreductase preferably to a derivative of such an enzyme for example a 5T4scFv fusion protein that can be secreted from the cell of production.
  • the resulting fusion protein is taken up by surrounding cells so increasing the number of cells capable of activating a prodrug.
  • the secretion signal is not necessarily associated with a targeting ligand such as an scFv and any suitable secretion signal sequence can be used. The addition of a secretion signal allows release of the fusion protein from the production cell in situations where the cell does not, naturally or as a consequence of cell death, liberate proteins.
  • Biochemical association domain In a third embodiment the localisation domain is a biochemical association domain.
  • biochemical association domain we mean a second prodrug activation domain such that products of one prodrug activation domain are delivered to another prodrug activation domain.
  • additional domain may be another enzyme such that the products of the first enzyme are directly delivered to a second enzyme. This achieves biochemical association that may or may not be combined with nuclear and trans-cellular targeting.
  • An example of biochemical association is where cytochrome
  • P450 reductase donates electrons to cytochrome P450 and the biochemical reaction is enhanced by proximity. Making a fusion between the two proteins can increase proximity so enhancing the efficiency of electron transfer to adjacent protein domains. This is useful, for example, for the activation of Cyclophosphamide which can be enhanced by the provision of excess cytochrome P450 (e.g. Chen et al 1996, Cancer res. 56, 1331; Patent Publication No.
  • prodrugs might be metabolised to different products depending upon the prodrug activation domains involved in the transformation or combinations of prodrug activation domains might potentiate the metabolism of a prodrug.
  • prodrug activation domains include Tirapazamine (Walton et al 1992 op. cit., J.M. Brown ip. cit.).
  • P450 reductase contribute to the hypoxic toxicity of Tirapazamine (Walton et al 1992 op. cit., J.M. Brown ip. cit.).
  • P450 reductase contribute to the hypoxic toxicity of Tirapazamine (Walton et al 1992 op. cit., J.M. Brown ip. cit.).
  • P450 reductase contribute to the hypoxic toxicity of Tirapazamine (Walton et al 1992 op. cit., J.M. Brown ip. cit.).
  • P450 reductase contribute to the hypo
  • the configuration of the combination will be dictated by the particular prodrug combination.
  • a number of configurations are outlined below by way of example:-
  • the coding region for P450 is fused to the coding region for P450 reductase or to one of the derivatives of P450R as outlined above. Fusions between a cytochrome P450 and P450 reductase have been described previously but not with the specific coding sequences described herein and not in the context of a gene therapy application (Blake et al 1996 FEBS Lett 397:210; cytochrome P450 3A4 fused to P450 reductase, M.S. Shet et al 1993 Proc. Natl. Acad. Sci.
  • the biochemical association domain may also be in the form of a macrophage.
  • the macrophage will express the prodrug activating enzyme.
  • P450 reductase is present in human macrophages.
  • P450 reductase is present in human macrophages.
  • P450 is not expressed in macrophages, but we have also found that macrophages can be engineered to express P450.
  • engineered macrophages can be used to activate prodrugs, such as cyclophosphamide. Other examples of prodrugs are given elsewhere in this description.
  • the macrophages are engineered to express the gene encoding the prodrug activating enzyme constitutively or alternatively regulatively.
  • suitable promoters are the cytomegalovirus CMV promoter and hypoxia response element (HRE); although other promoters may be used and will be known to those skilled in the art.
  • the prodrug activating agent which may be expressed by the macrophage is not limited to P450.
  • Other prodrug activating agents may be used such as cytosine deaminase. Such agents will be known to those skilled in the art.
  • a macrophage capable of expressing P450 Preferably the P450 is CYP2B6.
  • the P450 is under the control of a CMV promoter.
  • Such a prodrug activating agent may be used in therapy in combination with the prodrug cyclophosphamide.
  • Macrophages may also be used a method of delivery as will be described later.
  • the localisation domain is a chemical association domain.
  • chemical association domain we mean a domain which alters the intracellular chemical environment to optimse conditions for the activity of the prodrug activation domain.
  • bioreductive activation of certain prodrugs is dependent upon the hypoxic environment.
  • drugs such as Tirapazamine where the activation is reversed by molecular oxygen (reviewed by J.M. Brown Br. J. Cancer 1993, 67, 1163).
  • T apa___unine is activated by NADPH dependent reductases such as P450 reductase.
  • the additional protein is used to deplete the cell of molecular oxygen such that conditions for bioreduction of Tkapazamine are favoured.
  • cytochrome P450 enzymes with P450R.
  • the cytochrome P450 is a mono- oxygenase and will reduce molecular oxygen so increasing the hypoxic environment for P450 reductase to activate Tirapazamine. Enhancement of the activity of cytochrome P450 by P450 reductase has been proposed in microbial systems but the enhancement of P450 reductase activity by cytochrome P450 has not been proposed previously in any context.
  • the chemical association domain may also be used in concert with an intracellular, transcellular or biochemical association domain.
  • P450 utilises molecular oxygen it can be used to further potentiate the activation of drugs such as Tirapazamine (TPZ) and MMC by helping to maintain the hypoxic environment. It has been shown that co-adminstration of drugs such as Tirapazamine (TPZ) and MMC by helping to maintain the hypoxic environment. It has been shown that co-adminstration of TPZ and MMC.
  • cytochrome P450 and P450 reductase results in an enhancement of cell sensitivity to Thapazamine.
  • a further advantage of the combination of cytochrome P450 and P450 reductase is that it has utility for combination drug therapy with Cyclophosphamide and Tirapazamine.
  • prodrug activating enzymes and derivatives are as follows: for example the fusion protein 450FN can be co-expressed with P450 reductase or derivatives to potentiate CP activation and Tirapazamine activation.
  • Other drug combinations include
  • Mitochondrial DNA is a highly susceptible target for DNA adduct formation.
  • the mt-DNA genome is saturated by functional and essential coding sequences (94.4%) with very little redundancy, mt-DNA lesions are potentially more cytotoxic than nuclear DNA lesions since the probability of generating damage in a critical coding region is far greater.
  • mt-DNA has no histones to protect the DNA from attack, so it is highly susceptible to alkylation by an active metabolite or a radical-generating strand- breaking agent. Mitochondria have been shown to be an important target for agents such as MMC (Pritsos et al 1997 Oncol Res 6-7, 333).
  • a first step to enhance the delivery of a prodrug metabolite to mitochondrial DNA is to import the prodrug activating enzyme into the mitochondrion.
  • Mitochondrial import pathways are well described.
  • Mitochondrial proteins are synthesised as preprotein precursors containing an amino-terminal presequence. Few proteins are localised to mitochondria without such ammo-terminal extensions. These presequences are necessary and in some cases sufficient for import (Verner and Schatz 1988 Science 241, 1307; Fenton 1995 Amer J Human Genetics 57, 235).
  • presequences lack primary sequence homology, they are enriched for both basic and hydroxylated amino acids, lacking both acidic amino acids and long hydrophobic stretches, and may be able to form amphiphilic ⁇ -helical and ⁇ sheet conformations.
  • presequences lack primary sequence homology, they are enriched for both basic and hydroxylated amino acids, lacking both acidic amino acids and long hydrophobic stretches, and may be able to form amphiphilic ⁇ -helical and ⁇ sheet conformations.
  • DNA sequence encoding a mitochondrial import signal is fused to the coding sequence for the prodrug activating enzyme. Suitable import sequences are described in Vemer and Schatz 1988 and Fenton 1995.
  • the fusion protein is expressed in the target cell and the fusion protein is imported into the mitochondria.
  • HSCs HAEMATOPOEITIC STEM CELLS
  • HSCs a reservoir of embryonic cells
  • HSCs in mammals, are found within the fetal liver, spleen and bone marrow but after birth and throughout adult life, they are normally found only in the bone marrow. 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.
  • HSCs Erythroid and Megakaryocytic cells
  • Myeloid granulocytes and mononuclear phagocytes
  • Lymphoid lymphoid lineages
  • CFU colony forming unit
  • G-CSF granulocyte colony stimulating factor
  • Erthropoietin (EPO) and thrombopoietin (TPO) are structurally similiar cytokines and support respectively, the proliferation and differentiation for erythroid and megakaryocytic lineages as well as more primitive progenitors (Gotoh et al 1997 Ann Hematol 75: 207-213). TPO initiates its biologic effects by binding to the Mpl receptor, which is a member of the haematopoietic receptor family (Broudy et al 1997 Blood 89: 1896-1904). HOXB4 has been shown to be an important regulator of very early but not late haematopoietic cell proliferation (Sauvageau et al 1995 Genes Dev 9: 1753-1765).
  • the soluble Kit ligand proteins act as a ligand for the transmembrane tyrosine kinase receptor C-kit and stimulate mast cell and erythroid progenitors (91EP-810609).
  • the interleukins include IL-1, 11-2, IL-3, IL-4, IL-5, 11-6 and EL-12, which are capable of activating HSCs (see "Molecular biology and Biotechnology Ed RA Meyers 1995
  • mediators which are important in the positive regulation of haemopoiesis, are derived mainly from stromal cells in the bone marrow, but they are also produced by mature forms of differentiated myeloid and lymphoid cells.
  • SCF stem cell factor
  • the CFU-GM cell is the precursor of both neutrophils and mononuclear phagocytes. As the CFU-GM differentiates along the neutrophil pathway, several distinct morphological stages are seen. Myeloblast develop into promyelocytes and myelocytes, which mature and are released into the circulation as neutrophils. The one-way differentiation of cells from the CFU-GM into mature neutrophils is probably the result of acquiring specific growth/differentiation factor receptors at different stages of development.
  • MHC class II molecules and CD38 are expressed on the CFU-GM but not on mature neutrophils.
  • Other surface molecules acquired during the differentiation process include CD 13, CD 14 at low density, CDI 5, the ⁇ t integrin, VLA- 4, the ⁇ 2 integrins CDI la, b and c associated with CD 18 ⁇ 2 chains, complement receptors and CD 16 Fc ⁇ receptors.
  • CFU-GMs taking the monocyte pathway give rise initially to profilerating monoblasts.
  • Circulating monocytes are thought to be a replacement pool for tissue-resident macrophages.
  • the different forms of macrophages comprise the reticulo-endothelial system.
  • monocytes and macrophages Like mature neutrophils, mature monocytes and macrophages lose CD34. However, unlike neutrophils, they continue to express significant levels of MHC class II molecules. These molecules are clearly important for the presentation of antigen to T cells. Monocytes also acquire many of the same surface molecules as mature neutrophils.
  • APCs antigen-presenting cells
  • follicular dentritic cells follicular dentritic cells
  • Langerhans' cells interdigitating cells
  • HSCs In the first stage of differentiation into colony forming cells (such as CFU-GEMM) the HSCs express CD33 and CD34.
  • HSCs can usually be characterised by the presence of the cell glycoprotein CD34 (and possibly CD33) at the cell surface.
  • the vital burst forming units-erythroid (BFUE) cells of the erythroid lineage carry antigens CD33 and CD34 but these antigens are lost in later differentiation.
  • the myelomonocytic lineage which includes CFU-GM cells carry CD33 but not CD34 and this CD33 is subsequently lost.
  • the megakaryotic lineage leads initially to CFU Mega cells which carry CD34 which is also subsequently lost.
  • MHC major histocompatibility complex
  • the domains are fused by constructing a hybrid DNA molecule.
  • the hybrid molecule specifies the amino acid sequence of the additional molecule covalently linked to a DNA sequence that specifies all, or an active part, of the prodrug activation domain. If co-administration rather than fusion is required then two genes encoding the prodrug activation domain and the additional protein are incorporated into a suitable gene delivery vector (GDV) designed to ensure co-adminstration to a cell.
  • GDV gene delivery vector
  • the additional protein can be expressed from a separate expression cassette in the same vector by using a distinct promoter / enhancer, alternatively the inclusion of two coding sequences separated by an internal ribosome entry site IRES will achieve co-administration.
  • the invention provides a nucleic acid encoding the at least one component of the agent described herein, said nucleic acid capable of expressing the at least one component of the agent in a target cell.
  • the invention provides a nucleic acid encoding the fusion protein described herein, said nucleic acid capable of expressing the fusion protein in a target cell.
  • the invention provides a nucleic acid vector containing the nucleic acid encoding the at least one component of the system, said vector capable of delivering the nucleic acid to a target cell.
  • the invention provides a nucleic acid vector containing the nucleic acid encoding the fusion protein, said vector capable of delivering the nucleic acid to a target cell.
  • Suitable vectors according to the invention include viral vectors, in particular retroviral vectors.
  • the invention also provides nucleic acid encoding the fusion proteins of the invention. These may be constructed using standard recombinant DNA methodologies.
  • the nucleic acid may be RNA or DNA and is preferably DNA. Where it is RNA, manipulations may be performed via cDNA intermediates.
  • a nucleic acid sequence encoding the first region will be prepared and suitable restriction sites provided at the 5' and/or 3' ends. Conveniently the sequence is manipulated in a standard laboratory vector, such as a plasmid vector based on pBR322 or pUC19 (see below). Reference may be made to Molecular Cloning by Sambrook et al. (Cold Spring Harbor, 1989) or similar standard reference books for exact details of the appropriate techniques.
  • Nucleic acid encoding the second region may likewise be provided in a similar vector system. Sources of nucleic acid may be ascertained by reference to published literature or databanks such as Genbank.
  • vector or plasmid refers to discrete elements that are used to introduce heterologous DNA into cells for either expression or replication thereof. Selection and use of such vehicles are well within the skill of the artisan. Many vectors are available, and selection of appropriate vector will depend on the intended use of the vector, i.e. whether it is to be used for DNA amplification or for DNA expression, the size of the
  • DNA to be inserted into the vector and the host cell to be transformed with the vector.
  • Each vector contains various components depending on its function (amplification of DNA or expression of DNA) and the host cell for which it is compatible.
  • the vector components generally include, but are not limited to, one or more of the following: an origin of replication, one or more marker genes, an enhancer element, a promoter, a transcription termination sequence and a signal sequence.
  • An expression vector includes any vector capable of expressing nucleic acids that are operatively linked with regulatory sequences, such as promoter regions, that are capable of expression of such DNAs.
  • an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector, that upon introduction into an appropriate host cell, results in expression of the cloned DNA.
  • Appropriate expression vectors are well known to those with ordinary skill in the art and include those that are replicable in eukaryotic and/or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome.
  • DNAs encoding the fusion protein according to the invention may be inserted into a vector suitable for expression of cDNAs in mammalian cells, e.g. a CMV enhancer- based vector such as pEVRF (Matthias, et al. , ( 1989) NAR 17, 6418).
  • a CMV enhancer- based vector such as pEVRF (Matthias, et al. , ( 1989) NAR 17, 6418).
  • Plasmids employs conventional ligation techniques. Isolated plasmids or DNA fragments are cleaved, tailored, and religated in the form desired to generate the plasmids required. If desired, analysis to confirm correct sequences in the constructed plasmids is performed in a known fashion. Suitable methods for constructing expression vectors, preparing in vitro transcripts, introducing DNA into host cells, and performing analyses for assessing expression and function are known to those skilled in the art.
  • Gene presence, amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA, dot blotting (DNA or RNA analysis), or in situ hybridisation, using an appropriately labelled probe based on a sequence provided herein. Those skilled in the art will readily envisage how these methods may be modified, if desired.
  • the hybrid DNA molecule may be delivered to the target cell population by any suitable Gene Delivery Vehicle, GDV.
  • GDV Gene Delivery Vehicle
  • the GDV can be designed by a person ordinarily skilled in the art of recombinant DNA technology and gene expression to express the fusion protein at appropriate levels and with the cellular specificity demanded by a particular application.
  • the novel prodrug activating enzyme combinations are delivered by cells such as monocytes, macrophages, lymphocytes or hematopoietic stem cells.
  • a novel cell-dependent delivery system is used. In this system the genes encoding the prodrug activating proteins and protein combinations are introduced into a macrophage, monocyte or monocyte stem cell precursor ex vivo and then introduced into the patient.
  • a vector is a tool that allows or faciliates the transfer of an entity from one environment to another.
  • some vectors used in recombinant DNA techniques allow entities, such as a segment of DNA (such as a heterologous DNA segment, such as a heterologous cDNA segment), to be transferred into a target cell.
  • the vector may then serve to maintain the heterologous DNA within the cell or may act as a unit of DNA replication.
  • examples of vectors used in recombinant DNA techniques include plasmids, chromosomes, artificial chromosomes or viruses.
  • the vector can be delivered by viral or non- viral techniques.
  • Non-viral delivery systems include but are not limted to DNA transfection methods.
  • 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) (Nature Biotechnology 1996 14; 556), multivalent cations such as spermine, cationic lipids or polylysine, 1, 2,-bis (oleoyloxy)-3-(trimethylammoiiio) propane (DOTAP)-cholesterol complexes (Wolff and Trubetskoy 1998 Nature Biotechnology 16: 421) and combinations thereof.
  • CFAs cationic facial amphiphiles
  • DOTAP 1, 2,-bis (oleoyloxy)-3-(trimethylammoiiio) propane
  • DOTAP 1, 2,-bis (oleoyloxy)-3-(trimethylammoiiio) propane
  • DOTAP 1, 2,-bis (oleoyloxy)-3
  • Viral delivery systems include but are not limited to adenovirus vector, an adeno- associated viral (AAV) vector, a herpes viral vector, a retroviral vector, a lentiviral vector, a poxvirus, a pox-associated viral, a pox-lentiviral or a baculoviral vector.
  • AAV adeno-associated viral
  • retroviruses include but are not limited to: murine leukemia virus (MLV), human immunodeficiency virus (HIV), equine infectious anaemia virus (EIAV), mouse mammary tumour virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), Avian myelocytomatosis virus-29 (MC29), and Avian erythroblastosis virus (AEV).
  • MMV murine leukemia virus
  • HCV human immunodeficiency virus
  • EIAV equine infectious anaemia virus
  • MMTV mouse mammary tumour virus
  • RSV Rous sarcoma virus
  • FuSV Fujinami sarcoma virus
  • retroviruses 1997 Cold Spring Harbour Laboratory Press Eds: JM Coffm, SM Hughes, HE Varmus pp 758- 763).
  • Retroviruses may be broadly divided into two categories: namely, "simple” and "complex". Retroviruses may even be further divided into seven groups. Five of these groups represent retroviruses with oncogenic potential. The remaining two groups are the lentiviruses and the spumaviruses. A review of these retroviruses is presented in Coffin et al, 1991 (ibid).
  • the lentivirus group can be split even further into “primate” and "non-primate”.
  • primate lentiviruses include human immunodeficiency virus (HIV), the causative agent of human auto-immunodeficiency syndrome (AIDS), and simian immunodeficiency virus (SIV).
  • the non-primate lentiviral group includes the prototype "slow virus” visna/maedi virus (VMV), as well as the related caprine arthritis- encephalitis virus (CAEV), equine infectious anaemia virus (EIAV) and the more recently described feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV).
  • lentivirus family and other types of retroviruses are that lentiviruses have the capability to infect both dividing and non-dividing cells.
  • retroviruses - such as MLV - are unable to infect non-dividing cells such as those that make up, for example, muscle, brain, lung and liver tissue.
  • Each retroviral genome comprises genes called gag, pol and e «v which code for virion proteins and enzymes.
  • these genes are flanked at both ends by regions called long terminal repeats (LTRs).
  • LTRs are responsible for proviral integration, and transcription. LTRs also serve as enhancer-promoter sequences and can control the expression of the viral genes. Encapsidation of the retroviral RNAs occurs by virtue of apsi sequence located at the 5' end of the viral genome.
  • the LTRs themselves are identical sequences that can be divided into three elements, which are called U3, R and U5.
  • U3 is derived from the sequence unique to the 3' end of the RNA.
  • R is derived from a sequence repeated at both ends of the RNA and
  • U5 is derived from the sequence unique to the 5' end of the RNA.
  • the sizes of the three elements can vary considerably among different retroviruses.
  • the basic molecular organisation of an infectious retroviral RNA genome is (5') R - U5 - gag, pol, env - U3-R (3').
  • gag, pol and e «v may be absent or not functional.
  • the R regions at both ends of the RNA are repeated sequences.
  • U5 and U3 represent unique sequences at the 5' and 3' ends of the RNA genome respectively.
  • MLV tissue tropism
  • a particular MLV that has an envelope protein called 4070A is known as an amphotropic virus, and this can also infect human cells because its envelope protein "docks" with a phosphate transport protein that is conserved between man and mouse. This transporter is ubiquitous and so these viruses are capable of infecting many cell types.
  • env gene replacement with a heterologous env gene is an example of a technique or strategy used to target specifically certain cell types. This technique is called pseudotyping and examples may be found in WO-A- 98/05759, WO-A-98/05754, WO-A-97/17457, WO-A-96/09400 and WO-A-91/00047.
  • RRV recombinant retroviral vector
  • RRV refers to a vector with sufficient retroviral genetic information to allow packaging of an RNA genome, in the presence of packaging components, into a viral particle capable of infecting a target cell. Infection of the target cell includes reverse transcription and integration into the target cell genome.
  • the RRV in use typically carries non-viral coding sequences which are to be delivered by the vector to the target cell.
  • An RRV is incapable of independent replication to produce infectious retroviral particles within the final target cell.
  • at least part of one or more of the gag, pol and env protein coding regions essential for replication may be removed from the virus. This makes the retroviral vector replication-defective.
  • the removed portions may even be replaced by an NOI to generate a virus capable of integrating its genome into a host genome but wherein the modified viral genome is unable to propagate itself due to a lack of structural proteins.
  • expression of the NOI occurs - resulting in, for example, a therapeutic and/or a diagnostic effect.
  • the transfer of an NOI into a site of interest is typically achieved by: integrating the NOI into the recombinant viral vector; packaging the modified viral vector into a virion coat; and allowing transduction of a site of interest - such as a targeted cell or a targeted cell population.
  • Replication-defective retroviral vectors are typically propagated, for example to prepare suitable titres of the retroviral vector for subsequent transduction, by using a combination of a packaging or helper cell line and the recombinant vector. That is to say, that the three packaging proteins can be provided in trans.
  • a "packaging cell line” contains one or more of the retroviral gag, pol and env genes.
  • the packaging cell line produces the proteins required for packaging retroviral DNA but it cannot bring about encapsidation due to the lack of a psi region.
  • the helper proteins can package the /wz ' -positive recombinant vector to produce the recombinant virus stock. This virus stock can be used to transduce cells to introduce the NOI into the genome of the target cells.
  • psi packaging signal that contains additional sequences spanning from upstream of the splice donor to downstream of the gag start codon (Bender et al., 1987) since this has been shown to increase viral titres.
  • the recombinant virus whose genome lacks all genes required to make viral proteins can tranduce only once and cannot propagate.
  • These viral vectors which are only capable of a single round of transduction of target cells are known as replication defective vectors.
  • the NOI is introduced into the host/target cell genome without the generation of potentially harmful retrovirus.
  • Retroviral packaging cell lines in which the gag, pol and env viral coding regions are carried on separate expression plasmids that are independently transfected into a packaging cell line are preferably used.
  • This strategy sometimes referred to as the three plasmid transfection method (Soneoka et al., 1995), reduces the potential for production of a replication-competent virus since three recombinant events are required for wild type viral production.
  • As recombination is greatly facilitated by homology reducing or eliminating homology between the genomes of the vector and the helper can also be used to reduce the problem of replication-competent helper virus production.
  • transient transfected cell lines An alternative to stably transfected packaging cell lines is to use transient transfected cell lines.
  • Transient transfections may advantageously be used to measure levels of vector production when vectors are being developed. In this regard, transient transfection avoids the longer time required to generate stable vector-producing cell lines and may also be used if the vector or retroviral packaging components are toxic to cells.
  • Components typically used to generate retroviral vectors include a plasmid encoding the gag/pol proteins, a plasmid encoding the env protein and a plasmid containing an NOI. Vector production involves transient transfection of one or more of these components into cells containing the other required components.
  • the vector encodes toxic genes or genes that interfere with the replication of the host cell, such as inhibitors of the cell cycle or genes that induce apotosis, it may be difficult to generate stable vector-producing cell lines, but transient transfection can be used to produce the vector before the cells die. Also, cell lines have been developed using transient transfection that produce vector titre levels that are comparable to the levels obtained from stable vector-producing cell lines (Pear et al., 1993).
  • retroviral vectors In addition to manipulating the retroviral vector with a view to increasing vector titre, retroviral vectors have also been designed to induce the production of a specific NOI in transduced cells. As already mentioned, the most common retroviral vector design involves the replacement of retroviral sequences with one or more NOIs to create replication-defective vectors. With regard to regulation of expression of the NOI, there are three main approaches currently in use.
  • the simplest approach has been to use the promoter in the retroviral 5' LTR to control the expresssion of a cDNA encoding an NOI or to alter the enhancer/promoter of the LTR to provide tissue-specific expression or inducibility. Where multiple NOIs are inserted, the additional downsteam NOIs can be expressed from a polycistronic mRNA by the use of internal ribosome entry sites.
  • the NOI may be operably linked to an internal heterologous promoter. This arrangement permits more flexibility in promoter selection. Additional NOIs can still be expressed from the 5 'LTR or the LTR can be mutated to prevent expression following infection of a target cell.
  • the NOI is inserted together with regulatory control elements in the reverse orientation to the 5 'LTR.
  • Genomic sequences including enhancers, promoters, introns and 3' regions may be included. In this way it may be possible to achieve tightly regulated tissue-specific gene expression.
  • retroviral vectors in particular lentiviral vectors, as single transcription unit vectors whereby the HRE/promoter construct of the invention is placed within the 3 'LTR such that the resultant duplication of the 3 'LTR also leads to duplication of the regulatory sequence (i.e. the 5 'LTR of the provirus will contain the HRE/promoter construct duplicated from the 3 'LTR).
  • this arrangement enhances the activated response to hypoxia in a synergistic manner. Consequently, it is preferred to use a retroviral vector which comprises an HRE/promoter of the invention within its
  • the HRE/promoter construct (optionally together with any additional regulatory sequences such as tissue-specific enhancer elements) may be inserted into the 3' U3 region of the retroviral vector or the 5' U5 region, most preferably the 3' U3 region.
  • the NOI is not also inserted into the LTR since the resulting two copies in the provirus can decrease the size of the NOI which can be accommodated by the retroviral vector.
  • the NOI is preferably inserted into the region of the retroviral vector which is normally occupied by the env gene.
  • the NOI may or may not include a selectable marker. If the vector contains an NOI that is not a selectable marker, the vector can be introduced into packaging cells by co- transfection with a selectable marker present on a separate plasmid. This strategy has an appealing advantage for gene therapy in that a single protein is expressed in the ultimate target cells and possible toxicity or antigenicity of a selectable marker is avoided. However, when the inserted gene is not selectable, this approach has the disadvantage that it is more difficult to generate cells that produce a high titre vector stock. In addition it is usually more difficult to determine the titre of the vector.
  • the current methodologies used to design retroviral vectors that express two or more proteins have relied on three general strategies. These include: (i) the expression of different proteins from alternatively spliced mRNAs transcribed from one promoter; (ii) the use of the promoter in the 5' LTR and internal promoters to drive transcription of different cDNAs and (iii) the use of internal ribosomal entry site (IRES) elements to allow translation of multiple coding regions from either a single mRNA or from fusion proteins that can then be expressed from an open reading frame.
  • Vectors containing internal heterologous promoters have been widely used to express multiple genes. An internal promoter makes it possible to exploit promoter/enhancer combinations other than the viral LTR for driving gene expression. Multiple internal promoters can be included in a retroviral vector and it has proved possible to express at least three different cDNAs each from its own promoter.
  • the lentivirus group can be into “primate” and "non-primate”.
  • primate lentiviruses include human immunodeficiency virus (HTV), the causative agent of human auto-immunodeficiency syndrome (AIDS), and simian immunodeficiency virus (SIV).
  • the non-primate lentiviral group includes the prototype "slow virus” visna maedi virus (VMV), as well as the related caprine arthritis-encephalitis virus (CAEV), equine infectious anaemia virus (EIAV) and the more recently described feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV).
  • lentivirus family and other types of retroviruses are that lentiviruses have the capability to infect both dividing and non-dividing cells.
  • retroviruses - such as MLV - are unable to infect non-dividing cells such as those that make up, for example, muscle, brain, lung and liver tissue.
  • lentiviral vectors may advantageously be used in the present invention since lentiviruses are capable of infecting a wide range of non-dividing cells, by contrast to certain other retroviruses that require cell division for stable integration.
  • lentiviral vectors are based on HTV, SIV or EIAV.
  • the simplest vectors constructed from HfV-1 have the complete HIV genome except for a deletion of part of the env coding region or replacement of the nef coding region.
  • these vectors express gag/pol and all of the accessory genes hence require only an envelope to produce infectious virus particles.
  • the accessory genes vif, vpr, vpu and nef are non-essential. More recently however vectors have been described that are efficient yet lack most or all of the accessory factors, for example
  • a lentiviral vector of the invention preferably lacks at least one accessory gene, more preferably all accessory genes.
  • HTV-based lentiviral vectors HIV 5 'LTR and leader, some gag coding region sequences (to supply packaging functions), a reporter cassette, the rev response element (RRE) and the 3 'LTR.
  • gag coding region sequences to supply packaging functions
  • RRE rev response element
  • 3 'LTR the 3 'LTR.
  • accessory gene products and envelope functions are supplied either from a single plasmid or from two or more co-transfected plasmids, or by co-infection of vector containing cells with
  • HTV HTV More recently the lentiviral vector configurations have been further refined. For example self inactivating HTV vectors have been produced where the HIV LTR is deleted to restrict expression to the internal cassette (Myoshi et ⁇ l., 1998).
  • the lentiviral vector of the present invention is a non- primate lentiviral vector, for example EIAV.
  • EIAV non- primate lentiviral vector
  • a minimal EIAV genome vector typically comprises a promoter and optionally an enhancer capable of directing expression of a retroviral vector genome comprising, in order, the following elements from an EIAV: part of the 5 'LTR containing an R-region and a U5 region; sequences from the 5 'untranslated region of the gag gene containing a functional packaging signal and a portion of the gag coding sequence; an insertion site for a gene of interest and a 3 'LTR from EIAV.
  • the 3' LTR may be a hybrid LTR containing at least the polypurine tract and R-U5 region from an EIAV and sequences from another source to replace all or part of the U3 region.
  • the R region in the 5' and 3' LTRs may be replaced.
  • WO-A-96/33623 describes a method for replacing both the U3 and R regions of retroviral vector genomes.
  • the portion of the gag gene contains less than the first 350 nucleotides of the gag coding region, more preferably only the first 150 or 109 nucleotides of the gag coding region, only about the first 109 nucleotides being especially preferred.
  • a hCMV-MIE promoter enhancer is used to direct expression of the retroviral RNA transcript.
  • the U3 region enhancer may, for example, be replaced by a hypoxia responsive enhancer (HRE) of the present invention and an SV40 or MLV promoter.
  • HRE hypoxia responsive enhancer
  • the minimal EIAV vector also lacks S2, Tat, Rev and dUTPase genes but may still be used in vector production or for transduction of dividing and non-dividing cells.
  • the viral genome may typically be packaged in cells by providing gagpol and env functions in trans.
  • DNA sequences encoding gagpol and env may be co-introduced into a cell along with the minimal vector as described above for retroviruses in general.
  • the gagpol sequence is of non-primate lentiviral origin. It is particularly advantageous to include the leader sequences between the end of the 5' LTR and the ATG start codon of gag upstream of the gagpol coding sequence to provide for maximum gagpol expression. It may also be desirable to include the Rev and/or RRE sequences although this is not essential and may be eliminated by codon optimisation of the EIAV gagpol.
  • RNA genomic and subgenomic-sized RNA molecules that are generated by RNA processing.
  • all RNA products serve as templates for the production of viral proteins.
  • the expression of the RNA products is achieved by a combination of RNA transcript splicing and ribosomal frameshifting during translation.
  • RNA transcript splicing is carried out in higher eukaryotic cells by nuclear RNA processing machinery (the spliceosome) that recognises short consensus sequences at the boundaries of the sequences to be spliced.
  • These consensus splice sites follow the so-called 'GT-AG rule' (although in the RNA sequence GT is of course GU).
  • the 5' site, or the splice donor (SD) has a highly conserved GT dinucleotide at the first exon- intron boundary and the 3' site, or the splice acceptor (SA), has a highly conserved AG dinucleotide at the second exon-intron boundary.
  • SA splice acceptor
  • the splicing process also depends on the presence of a sequence within the region to be spliced out called a branch site.
  • the branch site is usually lies between 18 to 40 nucleotides upstream of the 3' SA and is believed to identify the nearest SA as the correct splice site for connection to the 5' SD.
  • retroviral RNA Unlike most cellular rnRNAs, in which all introns are efficiently spliced, newly synthesised retroviral RNA must be diverted into two populations. One population remains unspliced to serve as the genomic RNA and the other population is spliced to provide subgenomic RNA.
  • one population of newly synthesised retroviral RNA remains unspliced to serve as the genomic RNA and as mRNA for gag and pol.
  • the other population is spliced, fusing the 5' portion of the genomic RNA to the downstream genes, most commonly env.
  • Splicing is achieved with the use of a splice donor positioned upstream of gag and a splice acceptor near the 3 ' terminus of pol.
  • the intron between the splice donor and splice acceptor that is removed by splicing contains the gag and pol genes. This splicing event creates the mRNA for envelope (Env) protein.
  • the splice donor is upstream of gag but in some viruses, such as ASLV, the splice donor is positioned a few codons into the gag gene resulting in a primary Env translation product that includes a few amino-terminal __mino acid residues in common with Gag.
  • the Env protein is synthesised on membrane-bound polyribosomes and transported by the cell's vesicular traffic to the plasma membrane, where it is incorporated into viral particles.
  • Complex retroviruses generate both singly and multiply spliced transcripts that encode not only the env gene products but also the sets of regulatory and accessory proteins unique to these viruses.
  • Compex retroviruses such as the lentiviruses, and especially
  • HTV provide striking examples of the complexity of alternative splicing that can occur during retroviral infection.
  • HIV-1 is capable of producing over 30 different mRNAs by sub-optimal splicing from primary genomic transcripts. This selection process appears to be regulated as mutations that disrupt competing splice acceptors can cause shifts in the splicing patterns and can affect viral infectivity.
  • a retroviral vector is constructed which comprises a first nucleotide sequence (NS) capable of yielding a functional splice donor site (FSDS) and a second NS capable of yielding a functional splice acceptor site (FSAS); wherein the first NS is downstream of the second NS and is present in the sequences within the 3' LTR (the U3 region) of the retroviral vector that are duplicated in the 5' LTR as a result of reverse transcription of the RNA form of the retroviral vector into the DNA form that integrates into a host cell genome.
  • NS nucleotide sequence
  • FSAS functional splice acceptor site
  • the first NS capable of yielding an FSDS is downstream of the second NS capable of yielding an FSAS, splicing will not occur in the retroviral vector using these sites.
  • the reverse-transcribed DNA form of the vector such as the integrated provirus
  • the first NS is now upstream of the second NS and thus a functional 5' splice donor - 3' splice acceptor pairing is generated. Consequently, when transcription occurs from the provirus, the resulting RNA is capable of being spliced to remove intervening sequences between the FSDS and the FSAS.
  • this process has several applications.
  • a preferred embodiment in which split-intron technology may preferably be used relates to a hybrid vector system (see above).
  • the retroviral genome delivered by the primary viral vector, such as an adenoviral vector, to a primary target celll is in a configuration such the the first NS is downstream of the second NS.
  • the viral particles packaged by the primary target cell may be used to infect a secondary target cell. In the secondary target cell, reverse transcription and integration of the viral genome will typically occur. This will result in a functional splice donor/splice acceptor configuration since the FSDS will then be located in the 5'
  • LTR i.e. upstream of the FSAS. Splicing may then occur to remove the sequences between the FSDS and FSAS.
  • the nucleotide sequence between the 5' LTR and the second NS in the retroviral vector that is found between the FSAS and FSDS in the provirus may contain a nucleic acid sequence of interest (NOI).
  • NOI may contain sequences that are essential for production of viral particles, for example env or gagpol sequences. Since the NOI will be spliced out from proviral transcripts, the provirus will not be able to give rise to viable viral particles. This will, for example, allow construction of adeno retroviral vectors that contain NOIs encoding viral components and gene products of interest, such as therapeutic polypeptides, that have the added safety feature of not being able to produce said viral components when the viral particles assembled in the primary target cells are introduced into secondary target cells.
  • a further embodiment allows the expression of an NOI in a secondary target cell and not a primary target cell.
  • removal of the intervening sequences between the FSDS and the FSAS in the proviral RNA transcript brings a regulatory sequence that is 5' to the FSDS in the proviral genome near to the 5' end of an NOI that was 3' to the FSAS in the proviral genome such that the regulatory sequence is now operably linked to the NOI and can direct transcription from the NOI whereas when the intervening sequences were present, the regulatory sequence was not operably linked to the NOI.
  • the regulatory sequence may be upstream of the NOI in the retroviral vector, in a preferred embodiment the regulatory sequence is upstream of the first NS in the retroviral vector, within the U3 region of the 3' LTR such that it is downstream of the NOI.
  • the regulatory sequence positioned 5' of the NOI.
  • NOIs which are positioned between the FSDS and the FSAS (and are therefore upstream of the second NS in the retroviral vector) are sequences that will typically be spliced out of the viral RNA transcript produced by the provirus in the secondary target cell.
  • Such NOIs may therefore include, but are not limited to, retroviral sequences required for assembly of retroviral particles in the primary cells. These include env sequences, gagpol sequences, sequences encoding essential accessory genes and/or packaging sequences.
  • NOIs which are positioned downstream of the FSAS in the proviral genome will therefore typically include, but are generally not limited to, sequences encoding gene products that it is desired to express in the secondary target cells, such as therapeutic products.
  • retroviral vector in the context of an adenoretroviral hybrid system, includes the retroviral genome sequences present in a primary adenoretroviral vector as well as the subsequent RNA genome produced in the primary target cells and subsequently packaged into viral particles.
  • split-intron vectors may required functional inactivation of existing functional splice donor sites, for example to prevent splicing in the retroviral RNA genome transcribed in the primary target cells. Typically, this may be achieved by mutating the GT dinucleotide consensus, for example to GC, using standard techniques.
  • Adenoviruses The adenovirus is a double-stranded, linear DNA virus that does not go through an RNA intermediate.
  • RNA intermediate There are over 50 different human serotypes of adenovirus divided into 6 subgroups based on the genetic sequence homology all of which exhibit comparable genetic organisation.
  • Human adenovirus group C serotypes 2 and 5 (with 95% sequence homology) are most commonly used in adenoviral vector systems and are normally associated with upper respiratory tract infections in the young.
  • the adenoviruses/adenoviral vectors of the invention may be of human or animal origin.
  • preferred adenoviruses are those classified in group C, in particular the adenoviruses of type 2 (Ad2), 5 (Ad5), 7 (Ad7) or 12 (Adl2). More preferably, it is an Ad2 or Ad5 adenovirus.
  • Ad2 or Ad5 adenovirus canine adenovirus, mouse adenovirus or an avian adenovirus such as CELO virus (Cotton et al, 1993) may be used.
  • adenoviruses of canine origin and especially the strains of the CAV2 adenoviruses [manhattan strain or A26/61 (ATCC VR-800) for example].
  • Other adenoviruses of animal origin include those cited in application WO-A- 94/26914 incorporated herein by reference.
  • the organisation of the adenovirus genome is similiar in all of the adenovirus groups and specific functions are generally positioned at identical locations for each serotype studied.
  • the genome of adenoviruses comprises an inverted terminal repeat (TTR) at each end, an encapsidation sequence (Psi), early genes and late genes.
  • TTR inverted terminal repeat
  • Psi encapsidation sequence
  • the main early genes have been classified into an array of intermediate early (El a), delayed early (Elb, E2a, E2b, E3 and E4), and intermediate regions (see diagram below). Among these, the genes contained in the El region in particular are necessary for viral propagation.
  • the main late genes are contained in the LI to L5 regions.
  • the genome of the Ad5 adenovirus has been completely sequenced and is available on a database (see particularly Genbank Accession No. M73260). Likewise, parts, or even all of other adenoviral genomes (such as Ad2, Ad7, Ad 12) have also been sequenced.
  • an adenovirus is typically modified so as to make it incapable of replicating in an infected cell.
  • constructs described in the prior art include adenoviruses deleted for the El region, essential for viral replication, into which are inserted the heterologous DNA sequences (Levrero et al., 1991; Gosh-Choudhury et al., 1986).
  • a heat-sensitive point mutation has been introduced into the tsl25 mutant, making it possible to inactivate the 72 kDa DNA- binding protein (DBP).
  • a recombinant adenoviral vector used in the invention comprises a deletion in the El region of its genome.
  • the El region comprises a deletion in the Ela and Elb regions.
  • the El region is inactivated by deletion of a PvuII-Bglll fragment stretching from nucleotide 454 to nucleotide 3328, in the Ad5 adenovirus sequence (Genbank Accession No. M73260).
  • the El region is inactivated by deletion of an Hi ⁇ /II-Sau3A fragment stretching from nucleotide 382 to nucleotide 3446.
  • adenoviral vectors comprise a deletion of another region essential for viral replication and/or propagation, the E4 region.
  • the E4 region is involved in the regulation of the expression of the late genes, in the stability of the late nuclear RNAs, in decreasing host cell protein expression and in the efficiency of the replication of the viral DNA.
  • Adenoviral vectors in which the El and E4 regions are deleted therefore possess very reduced viral gene expression and transcriptional background noise.
  • Such vectors have been described for example in applications WO-A-94/28152, WO-A- 95/02697, WO-A-96/22378.
  • vectors carrying a modification of the IVa2 gene have also been described (WO-A-96/10088).
  • a recombinant adenoviral vector used in the invention comprises, in addition, a deletion in the E4 region of its genome. More particularly, the deletion in the E4 region affects all the open reading frames. There may be mentioned, by way of a precise example, deletions of nucleotides 33466-35535 or 33093-35535.
  • preferred vectors comprise a deletion of the whole of the E4 region. This may be carried deletion or excision of an Maell-Mscl fragment corresponding to nucleotides
  • ORF3 and ORF6 frames can be deleted from the genome in the form of PvuII-AluI and BgUI-PvuII fragments respectively, corresponding to nucleotides 34801-34329 and 34115-33126 respectively.
  • the deletions of the E4 region of the virus Ad2 dl808 or of viruses Ad5 dll004, Ad5 dll007, Ad5 dllOl 1 or Ad5 dll014 can also be used within the framework of the invention.
  • positions given above refer to the wild-type Ad5 adenovirus sequence as published and accessible on a database. Although minor variations may exist between the various adenovirus serotypes, these positions are generally applicable to the construction of recombinant adenoviruses according to the invention from any serotype, and especially the adenoviruses Ad2 and Ad7.
  • the adenoviruses produced may possess other alterations in their genome.
  • other regions may be deleted to increase the capacity of the virus and reduce its side effects linked to the expression of viral genes.
  • all or part of the E3 or _Va2 region in particular may be deleted.
  • the E3 region it may however be particularly preferred to conserve the part encoding the gpl9K protein. This protein indeed makes it possible to prevent the adenoviral vector from becoming the subject of an immune reaction which (i) would limit its action and (ii) could have undesirable side effects.
  • the E3 region is deleted and the sequence encoding the gpl9K protein is reintroduced under the control of a heterologous promoter.
  • the polynucleotide of the invention/NOI can be inserted into various sites of the recombinant genome. It can be inserted at into the El, E3 or E4 region, as a replacement for the deleted or surplus sequences. It can also be inserted into any other site, outside the sequences necessary in cis for the production of the viruses (UK sequences and encapsidation sequence).
  • the E2 region is essential as it encodes the 72 kDa DNA binding protein, DNA polymerase and the 80 kDa precurser of the 55 kDa Terminal Protein (TP) needed for protein priming to initiate DNA synthesis.
  • TP Terminal Protein
  • the recombinant adenoviruses are typically produced in an encapsidation cell line, which is a cell line capable of complementing in trans one or more of the functions deficient in the recombinant adenoviral genome.
  • an encapsidation cell line which is a cell line capable of complementing in trans one or more of the functions deficient in the recombinant adenoviral genome.
  • One of these lines is for example line
  • 293 is a human kidney embryonic cell line containing the left end (about 11-12%) of the genome of serotype 5 adenovirus (Ad5), comprising the left ITR, the encapsidation region, the El region, including Ela and E lb, the region encoding protein pIX and part of the region encoding protein p_Va2.
  • This line is capable of transcomplementing recombinant adenoviruses defective for the El region, that is to say lacking all or part of the El region, and of producing viral stocks having high titres.
  • This line is also capable of producing, at a permissive temperature (32°C), virus stocks comprising, in addition, the heat-sensitive E2 mutation.
  • cell lines capable of complementing the El region have been described, based in particular on human lung carcinoma cells A549 (WO-A-94/28152) or on human retinoblasts (Hum. Gen. Ther. (1996) 215).
  • cell lines capable of transcomplementing several adenovirus functions have also been described, for example cell lines complementing the El and E4 regions (Yeh et al., 1996a, b; Krougliak et al, 1995) and lines complementing the El and E2 regions (WO-A-94/28152, WO-A-
  • the recombinant adenoviruses are usually produced by introducing the viral DNA into the encapsidation line, followed by lysis of the cells after about 2 or 3 days (the kinetics of the adenoviral cycle being 24 to 36 hours).
  • the viral DNA introduced may be the complete recombinant viral genome, optionally constructed in a bacterium (WO-A-96/25506) or in a yeast (WO-A-95/03400), transfected into the cells. It may also be a recombinant virus used to infect the encapsidation line.
  • the viral DNA may also be introduced in the form of fragments each carrying part of the recombinant viral genome and a region of homology which makes it possible, after introduction into the encapsidation cell, to reconstitute the recombinant viral genome by homologous recombination between the various fragments.
  • Replication-competent adenoviruses can also be used for gene therapy.
  • the Ela gene can be inserted into a first generation virus under the regulation of a tumour-specific promoter.
  • This type of vector could be used either to kill tumour cells directly by lysis or to deliver a "suicide gene" such as the herpes-simplex-virus thymidine-kinase gene (HSV tk) which can kill infected and bystander cells following treatment with ganciclovir.
  • a "suicide gene” such as the herpes-simplex-virus thymidine-kinase gene (HSV tk) which can kill infected and bystander cells following treatment with ganciclovir.
  • the viral genes may be placed under the control of a hypoxia responsive regulatory element.
  • Hybrid adenovirus/retrovirus systems may also be used whereby the features of adenoviruses are combined with the genetic stability of retroviruses.
  • These hybrid viral vectors use the adenovirus to transduce target cells when then become transient retroviral producer cells that can stably infect neighbouring cells.
  • a hybrid viral vector system of the present invention comprises one or more primary viral vectors which encode a secondary viral vector, the primary vector or vectors being capable of infecting a first target cell and of expressing therein the secondary viral vector, which secondary vector is capable of transducing a secondary target cell.
  • a genetic vector of the invention consists of a primary vector manufactured in vitro which encodes the genes necessary to produce a secondary vector in vivo.
  • the secondary vector carries one or more selected genes for insertion into the secondary target cell.
  • the selected genes may be one or more marker genes and/or therapeutic genes (see above).
  • the primary viral vector or vectors may be a variety of different viral vectors, such as retroviral (including lentiviral), adenoviral, herpes virus or pox virus vectors, or in the case of multiple primary viral vectors, they may be a mixture of vectors of different viral origin. In whichever case, the primary viral vectors are preferably defective in that they are incapable of independent replication. Thus, they are capable of entering a target cell and delivering the secondary vector sequences, but not of replicating so as to go on to infect further target cells.
  • retroviral including lentiviral
  • adenoviral adenoviral
  • herpes virus or pox virus vectors or in the case of multiple primary viral vectors, they may be a mixture of vectors of different viral origin.
  • the primary viral vectors are preferably defective in that they are incapable of independent replication. Thus, they are capable of entering a target cell and delivering the secondary vector sequences, but not of replicating so as to go on to infect further target cells.
  • the hybrid viral vector system comprises more than one primary vector to encode the secondary vector
  • all of the primary vectors will be used to infect a primary target cell population, usually simultaneously.
  • the preferred single or multiple primary viral vectors are adenoviral vectors.
  • Adenovirus vectors have significant advantages over other viral vectors in terms of the titres which can be obtained from in vitro cultures.
  • the adenoviral particles are also comparatively stable compared with those of enveloped viruses and are therefore more readily purified and stored.
  • the secondary viral vector is preferably a retroviral vector, more preferably a lentiviral vector.
  • the secondary vector is produced by expression of essential genes for assembly and packaging of a defective viral vector particle, within the primary target cells. It is defective in that it is incapable of independent replication. Thus, once the secondary retroviral vector has transduced a secondary target cell, it is incapable of spreading by replication to any further target cells.
  • the secondary vector may be produced from expression of essential genes for retroviral vector production encoded in the DNA of the primary vector.
  • genes may include a gag-pol gene from a retrovirus, an envelope gene from an enveloped virus and a defective retroviral genome containing one or more therapeutic genes.
  • the defective retroviral genome contains in general terms sequences to enable reverse transcription, at least part of a 5' long terminal repeat (LTR), at least part of a 3 'LTR and a packaging signal.
  • LTR 5' long terminal repeat
  • the secondary vector is also safe for in vivo use in that incorporated into it are one or more safety features which eliminate the possibility of recombination to produce an infectious virus capable of independent replication.
  • the secondary vector may be encoded by a plurality of transcription units, which may be located in a single or in two or more adenoviral or other primary vectors.
  • a transcription unit encoding the secondary vector genome there may be a transcription unit encoding gag-pol and a transcription unit encoding env.
  • two or more of these may be combined.
  • nucleic acid sequences encoding gag-pol and env, or env and the genome may be combined in a single transcription unit. Ways of achieving this are known in the art.
  • Transcription units as described herein are regions of nucleic acid containing coding sequences and the signals for achieving expression of those coding sequences independently of any other coding sequences.
  • each transcription unit generally comprises at least a promoter, an enhancer and a polyadenylation signal.
  • the promoter and enhancer of the transcription units encoding the secondary vector are preferably strongly active, or capable of being strongly induced, in the primary target cells under conditions for production of the secondary viral vector.
  • the promoter and/or enhancer may be constitutively efficient, or may be tissue or temporally restricted in their activity.
  • Safety features which may be incorporated into the hybrid viral vector system are described below. One or more such features may be present.
  • sequence homology between the sequences encoding the components of the secondary vector may be avoided by deletion of regions of homology. Regions of homology allow genetic recombination to occur.
  • three transcription units are used to construct a secondary retroviral vector.
  • a first transcription unit contains a retroviral gag-pol gene under the control of a non-retroviral promoter and enhancer.
  • a second transcription unit contains a retroviral env gene under the control of a non-retroviral promoter and enhancer.
  • a third transcription unit comprises a defective retroviral genome under the control of a non-retroviral promoter and enhancer.
  • the packaging signal is located such that part of the gag sequence is required for proper functioning.
  • the packaging signal including part of the gag gene, remains in the vector genome.
  • the defective retroviral genome contains a minimal packaging signal which does not contain sequences homologous to gag sequences in the first transcription unit.
  • retroviruses for example Moloney Murine Leukaemia virus (MMLV)
  • MMLV Moloney Murine Leukaemia virus
  • the corresponding region of homology between the first and second transcription units may be removed by altering the sequence of either the 3' end of the pol coding sequence or the 5' end of env so as to change the codon usage but not the amino acid sequence of the encoded proteins.
  • the retroviral vector is constructed from the following three components.
  • the first transcription unit contains a retroviral gag-pol gene under the control of a non-retroviral promoter and enhancer.
  • the second transcription unit contains the env gene from the alternative enveloped virus, under the control of a non-retroviral promoter and enhancer.
  • the third transcription unit comprises a defective retroviral genome under the control of a non- retroviral promoter and enhancer.
  • the defective retroviral genome contains a minimal packaging signal which does not contain sequences homologous to gag sequences in the first transcription unit.
  • Pseudotyping may involve for example a retroviral genome based on a lentivirus such as an HIV or equine infectious anaemia virus (EIAV) and the envelope protein may for example be the amphotropic envelope protein designated 4070A.
  • the retroviral genome may be based on MMLV and the envelope protein may be a protein from another virus which can be produced in non-toxic amounts within the primary target cell such as an Influenza haemagglutinin or vesicular stomatitis virus G protein.
  • the envelope protein may be a modified envelope protein such as a mutant or engineered envelope protein. Modifications may be made or selected to introduce targeting ability or to reduce toxicity or for another purpose.
  • the possibility of replication competent retroviruses can be eliminated by using two transcription units constructed in a particular way.
  • the first transcription unit contains a gag-pol coding region under the control of a promoter-enhancer active in the primary target cell such as a hCMV promoter-enhancer or a tissue restricted promoter- enhancer.
  • the second transcription unit encodes a retroviral genome RNA capable of being packaged into a retroviral particle.
  • the second transcription unit contains retroviral sequences necessary for packaging, integration and reverse transcription and also contains sequences coding for an env protein of an enveloped virus and the coding sequence of one or more therapeutic genes.
  • the hybrid viral vector system comprises single or multiple adenoviral primary vectors which encodes or encode a retroviral secondary vector.
  • Adenoviral vectors for use in the invention may be derived from a human adenovirus or an adenovirus which does not normally infect humans.
  • the vectors are derived from Adenovirus Type 2 or adenovirus Type 5 (Ad2 or Ad5) or a mouse adenovirus or an avian adenovirus such as CELO virus.
  • the vectors may be replication competent adenoviral vectors but are more preferably defective adenoviral vectors.
  • Adenoviral vectors may be rendered defective by deletion of one or more components necessary for replication of the virus.
  • each adenoviral vector contains at least a deletion in the El region.
  • this deletion may be complemented by passage of the virus in a human embryo fibroblast cell line such as human 293 cell line, containing an integrated copy of the left portion of Ad5, including the El gene.
  • the capacity for insertion of heterologous DNA into such vectors can be up to approximately 7 kb.
  • adenoviral vectors are known in the art which contain further deletions in other adenoviral genes and these vectors are also suitable for use in the invention.
  • Extended deletions serve to provide additional cloning capacity for the introduction of multiple genes in the vector. For example a 25 kb deletion has been described (Lieber et al, 1996) and a cloning vector deleted of all viral genes has been reported (Fisher et al, 1996) which will permit the introduction of more than 35 kb of heterologous DNA.
  • Such vectors may be used to generate an adenoviral primary vector according to the invention encoding two or three transcription units for construction of the retroviral secondary vector.
  • Embodiments of the invention described solve one of the major problems associated with adenoviral and other viral vectors, namely that gene expression from such vectors is transient.
  • the retroviral particles generated from the primary target cells can infect secondary target cells and gene expression in the secondary target cells is stably maintained because of the integration of the retroviral vector genome into the host cell genome.
  • the secondary target cells do not express significant amounts of viral protein antigens and so are less immunogenic than the cells transduced with adenoviral vector.
  • retroviral vector as the secondary vector is also advantageous because it allows a degree of cellular discrimination, for instance by permitting the targeting of rapidly dividing cells. Furthermore, retroviral integration permits the stable expression of therapeutic genes in the target tissue, including stable expression in proliferating target cells.
  • the primary viral vector preferentially infects a certain cell type or cell types. More preferably, the primary vector is a targeted vector, that is it has a tissue tropism which is altered compared to the native virus, so that the vector is targeted to particular cells.
  • the term “targeted vector” is not necessarily linked to the term “target cell”.
  • “Target cell” simply refers to a cell which a vector, whether native or targeted, is capable of infecting or transducing.
  • Primary target cells for the vector system according to the invention include but are not limited to haematopoietic cells (including monocytes, macrophages, lymphocytes, granulocytes or progenitor cells of any of these); endothelial cells; tumour cells; stromal cells; astrocytes or glial cells; muscle cells; and epithelial cells.
  • haematopoietic cells including monocytes, macrophages, lymphocytes, granulocytes or progenitor cells of any of these
  • endothelial cells including monocytes, macrophages, lymphocytes, granulocytes or progenitor cells of any of these
  • endothelial cells including monocytes, macrophages, lymphocytes, granulocytes or progenitor cells of any of these
  • endothelial cells including monocytes, macrophages, lymphocytes, granulocytes or progenitor cells of any of these
  • endothelial cells including tumour cells
  • a primary target cell according to the invention capable of producing the second viral vector, may be of any of the above cell types.
  • the primary target cell according to the invention is a monocyte or macrophage infected by a defective adenoviral vector containing a first transcription unit for a retroviral gag-pol and a second transcription unit capable of producing a packageable defective retroviral genome.
  • at least the second transcription unit is preferably under the control of a promoter-enhancer which is preferentially active in a diseased location within the body such as an ischaemic site or the micro-environment of a solid tumour.
  • the second transcription unit is constructed such that on insertion of the genome into the secondary target cell, an intron is generated which serves to reduce expression of the viral env gene and permit efficient expression of a therapeutic gene.
  • the secondary viral vectors may also be targeted vectors.
  • retroviral vectors this may be achieved by modifying the envelope protein.
  • the envelope protein of the retroviral secondary vector needs to be a non-toxic envelope or an envelope which may be produced in non-toxic amounts within the primary target cell, such as for example a MMLV amphotropic envelope or a modified amphotropic envelope.
  • the safety feature in such a case is preferably the deletion of regions or sequence homology between retroviral components.
  • the secondary target cell population may be the same as the primary target cell population.
  • delivery of a primary vector of the invention to tumour cells leads to replication and generation of further vector particles which can transduce further tumour cells.
  • the secondary target cell population may be different from the primary target cell population.
  • the primary target cells serve as an endogenous factory within the body of the treated individual and produce additional vector particles which can infect the secondary target cell population.
  • the primary target cell population may be haematopoietic cells transduced by the primary vector in vivo or ex vivo.
  • the primary target cells are then delivered to or migrate to a site within the body such as a tumour and produce the secondary vector particles, which are capable of transducing for example tumour cells within a solid tumour.
  • the invention permits the localised production of high titres of defective retroviral vector particles in vivo at or near the site at which action of a therapeutic protein or proteins is required with consequent efficient transduction of secondary target cells.
  • the invention also permits the production of retroviral vectors such as MMLV-based vectors in non-dividing and slowly-dividing cells in vivo. It had previously been possible to produce MMLV-based retroviral vectors only in rapidly dividing cells such as tissue culture-adapted cells proliferating in vitro or rapidly dividing tumour cells in vivo. Extending the range of cell types capable of producing retroviral vectors is advantageous for delivery of genes to the cells of solid tumours, many of which are dividing slowly, and for the use of non-dividing cells such as endothelial cells and cells of various haematopoietic lineages as endogenous factories for the production of therapeutic protein products.
  • inducible or regulated we mean that expression may be preferabltially activated, e.g. expression may be altered in response to extracellular cues.
  • constitutive expression we mean produced in substantially constant amount; opposite of regulated. Constitutive expression, e.g. occurs continuously without requiring an external stimulus.
  • the nucleic acid in a vector according to the invention is operatively linked to an expression control sequence capable of causing preferential expression of the fusion protein in the target cell.
  • the expression control sequence may be for example a promotor or enhancer which is preferentially active in certain cell types including the target cell, or a promotor or enhancer which is preferentially active under certain conditions such as hypoxic conditions.
  • Expression control sequences which are capable of causing preferential expression under conditions of hypoxia are known as hypoxia responsive elements (Dachs et al 1997 Nature Medicine 3, 515).
  • the nucleic acid may be placed under the control of a promoter that is specifically activated, for example by hypoxia, only when the macrophage has entered the tumour.
  • This aspect of the invention is particularly useful for those prodrugs, for example Tirapazamine, RSU1069, EO9 and Mitomycin C, that are themselves markedly activated in the conditions such as hypoxia that exist preferentially in tumours.
  • the nucleic acid may be under the expression control of an expression regulatory element, usually a promoter or a promoter and enhancer.
  • the enhancer and/or promoter may be preferentially active in a hypoxic or ischaemic or low glucose environment, such that the nucleic acid is preferentially expressed in the particular tissues of interest, such as in the environment of a tumour, arthritic joint or other sites of ischaemia.
  • tissue of interest such as in the environment of a tumour, arthritic joint or other sites of ischaemia.
  • the enhancer element or other elements conferring regulated expression may be present in multiple copies.
  • the enhancer and/or promoter may be preferentially active in one or more specific cell types - such as any one or more of macrophages, endothelial cells or combinations thereof.
  • 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 cess and post-mitotically terminally differentiated non-replicating cells such as macrophages neurons.
  • promoter is used in the normal sense of the art, e.g. an RNA polymerase binding site in the Jacob-Monod theory of gene expression.
  • the term “enhancer” includes a DNA sequence which binds to other protein components of the transcription initiation complex and thus facilitates the initiation of transcription directed by its associated promoter.
  • the promoters of the present invention are tissue specific. That is, they are capable of driving transcription of a nucleic acid in one tissue while remaining largely
  • tissue specific means a promoter which is not restricted in activity to a single tissue type but which nevertheless shows selectivity in that they may be active in one group of tissues and less active or silent in another group.
  • a desirable characteristic of the promoters of the present invention is that they posess a relatively low activity in the absence of activated hypoxia-regulated enhancer elements, even in the target tissue.
  • One means of achieving this is to use "silencer" elements which suppress the activity of a selected promoter in the absence of hypoxia.
  • tissue specific promoters may be particularly advantageous in practising the present invention.
  • these promoters may be isolated as convenient restriction digestion fragments suitable for cloning in a selected vector.
  • promoter fragments may be isolated using the polymerase chain reaction. Cloning of the amplified fragments may be facilitated by incorporating restriction sites at the 5' end of the primers.
  • Promoters suitable for cardiac-specific expression include the promoter from the murine cardiac ⁇ -myosin heavy chain (MHC) gene.
  • Suitable vascular endothelium-specific promoters include the Et-1 promoter and von Willebrand factor promoter.
  • Prostate specific promoters include the 5 'flanking resion of the human glandular kallikrein-1 (hKLK2) gene and the prostate specific antigen (hKLK3).
  • promoters/enhancers which are cell specific include a macrophage-specific promoter or enhancer, such as CSF-1 promoter-enhancer, or elements from a mannose receptor gene promoter-enhancer (Rouleux et al 1994 Exp Cell Res 214:113-119).
  • promoter or enhancer elements which are preferentially active in neutrophils, or a lymphocyte-specific enhancer such as an IL-2 gene enhancer, may be used.
  • HRE elements contain polynucleotide sequences that may be located either upstream (5') or downstream (3') of the promoter and/or therapeutic gene.
  • the HRE enhancer element (HREE) is typically a cw-acting element, usually about 10-300 bp in length, that acts on a promoter to increase the transcription of a gene under the control of the promoter.
  • the promoter and enhancer elements are selected such that expression of a gene regulated by those elements is minimal in the presence of a healthy supply of oxygen and is upregulated under hypoxic or anoxic conditions.
  • hypoxia means a condition under which a particular organ or tissue receives an inadequate supply of oxygen.
  • the hypoxia response element may also be selected from, for example, the erythropoietin HRE element (HREE1), muscle pyruvate kinase (PKM), HRE element, B-enolase (enolase 3; ENO3) HRE element, endothelin-1 (ET-l)HRE element and metallothionein ⁇ (MTU) HRE element.
  • HREE1 erythropoietin HRE element
  • PLM muscle pyruvate kinase
  • HRE element B-enolase (enolase 3; ENO3) HRE element
  • ENO3 endothelin-1
  • MTU metallothionein ⁇
  • hypoxia regulated enhancer is a binding element for the transcription factor HJF-1 (Dachs et al 1997 Nature Med 5: 515; Wang and Sememnza 1993 Proc Natl Acad Sci USA 90:4304; Firth et al 1994 Proc Natl Acad Sci USA 91 : 6496).
  • Hypoxia response enhancer elements have also been found in association with a number of genes including the erythropoietin (EPO) gene (Madan et al 1993 Proc Natl Acad Sci 90: 3928; Semenza and Wang 1992 Mol Cell Biol 1992 12: 5447-5454).
  • HREEs have been isolated from regulatory regions of both the muscle glycolytic enzyme pyrivate kinase (PKM) gene (Takenaka et al 1989 J Biol Chem 264: 2363- 2367), the human muscle-specific ⁇ -enolase gene (ENO3; Peshavaria and Day 1991 Biochem J 275: 427-433 ) and the endothelin-1 (ET-1) gene (Inoue et al 1989 J Biol
  • PLM muscle glycolytic enzyme pyrivate kinase
  • the promoter and/or enhancer should be constitutively efficient.
  • constitutive promoters such as cytomegalovirus (CMV) are known in the art and any may be used.
  • CMV cytomegalovirus
  • One prefened promoter-enhancer combination is a human cytomegalovirus (hCMV) major immediate early (ME) promoter/enhancer combination.
  • hCMV human cytomegalovirus
  • ME major immediate early
  • Polynucleic acid 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.
  • host cells comprising a polynucleic acid sequence 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 using by infection.
  • Target cells in 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.
  • 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.
  • the target cell may be a precursor of a differentiated cell such as a monocyte precursor, a CD33 + 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.
  • Preferred host cells/target cells include cells in which EPAS is expressed, more preferably cells in which EPAS is expressed but HEF-1 is not (or at much lower levels).
  • 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.
  • microenvironmental factors such as cell-to-cell interactions and the presence of soluble cell cytokines.
  • erythroid erythrocytes
  • megakaryocytic platelets
  • myeloid granulocytes and monocytes
  • lymphoid lymphoid
  • 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 conelated 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.
  • monocytes and macrophages are suitable host cells for use in the present invention.
  • 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.
  • the vector is preferably a targeted vector capable of targeting CD34+ HSCs.
  • target vector refers to a vector whose ability to infect/transfect a cell or to be expressed in the target cell is restricted to certain cell types within the host organism, usually cells having a common or similar phenotype.
  • An example of a targeted vector is a targeted retroviral vector with a genetically modified envelope protein which binds to cell surface molecules found only on a limited number of cell types in the host organism.
  • a targeted vector is one which contains promoter and/or enhancer elements which only permit expression of one or more retroviral transcripts in a proportion of the cell types of the host organism.
  • the vector may be provided with a ligand specific for CD34, such as an antibody or an immunoglobulin-like molecule directed against CD34.
  • a ligand specific for CD34 such as an antibody or an immunoglobulin-like molecule directed against CD34.
  • the vector may be administered systemically, to the peripheral circulation.
  • the present invention is believed to have a wide therapeutic applicability - depending on inter alia the selection of the one or more NOIs, prodrug activating domains and/or prodrugs.
  • the present invention may be useful in the treatment of disorders listed in WO-A-98/09985.
  • 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-
  • retmitis or cystoid macular oedema sympathetic ophthalmia, scleritis, retmitis 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.
  • monocyte or leukocyte prohferative diseases e.g. leukaemia
  • monocytes or lymphocytes 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.
  • tumours examples 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.
  • 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
  • nucleic acid sequencs may be expressed in hypoxic cells and not cells under normoxic conditions.
  • nucleic acid sequences, 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.
  • 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.
  • nucleic acid sequences, 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.
  • nucleic acid sequences, nucleic acid vectors, viral vectors and host cells of the present invention may also be used in preventative medicine.
  • the prodrugs used in the invention may have a therapeutic effect via prophylaxis.
  • 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 (Comelisse et al, 1996) or another relevant gene.
  • nucleic acid sequences, 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.
  • nucleic acid sequences 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.
  • nucleic acid sequences may be administered as part of a nucleic acid vector, including a plasmid vector or viral vector, preferably a lentiviral vector.
  • the delivery vehicle i.e. naked nucleic acid construct or viral vector comprising the polynucleotide for example
  • a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical composition.
  • 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 nucleic acid sequences, 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.
  • 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.
  • 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.
  • 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.
  • 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 nucleic acid sequences/vector containing the therapeutic gene for gene therapy, can be incorporated into cells at an appropriate area.
  • compositions may comprise from about 0.1% to about 99% by weight of the formulation.
  • pharmaceutically acceptable is meant that the ingredient must be compatible with other ingredients of the compositions as well as physiologically acceptable to the patient.
  • compositions for use according to the present invention may be formulated in conventional manner using readily available pharmaceutical or veterinary aids.
  • the active ingredient may be incorporated, optionally together with other active substances, with one or more conventional carriers, diluents and/or excipients, to produce conventional galenic preparations such as tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft and hard gelatin capsules, suppositories, sterile injectable solutions, sterile packaged powders, and the like.
  • Suitable carriers, excipients, and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, aglinates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water syrup water, water/ethanol, water/glycol, water/polyethylene, glycol, propylene glycol, methyl cellulose, methylhydroxybenzoates, propyl hydroxybenzoates, talc, magnesium stearate, mineral oil or fatty substances such as hard fat or suitable mixtures thereof.
  • compositions may additionally include lubricating agents, wetting agents, emulsifying agents, suspending agents, preserving agents, sweetening agents, flavouring agents, and the like.
  • the formulations may be formulated so as to provide quick, sustained or delayed release of the active ingredient after adminstration to the patient by use of procedures well known in the art.
  • the compositions are preferably formulated in a unit dosage form, e.g. with each dosage containing from about 0.1 to about 500 mg of the active ingredient.
  • an effective dose will be of the order of from about 0.01 mg/kg to about 20 mg/kg bodyweight per day, e.g. from about 0.05 to about 10 mg/kg per day, administered one or more times daily.
  • an appropriate dose for an adult may be from 10 to 100 mg per day, e.g. 20 to 50 mg per day.
  • Administration may be by any suitable method known in the art, including for example oral, parenteral (e.g. intramuscular, subcutaneous, intraperitoneal or intravenous), rectal or topical administration.
  • parenteral e.g. intramuscular, subcutaneous, intraperitoneal or intravenous
  • rectal or topical administration e.g., rectal or topical administration.
  • nucleic acid sequences of the invention is delivered to cells by a viral vector
  • the amount of virus administered is in the range of from 10 to 10
  • the amount of nucleic acid administered is typically in the range of from 1 ⁇ g to 10 mg, preferably from 100 ⁇ g to 1 mg.
  • NOI is under the control of an inducible regulatory sequence
  • the inducer is removed and expression of the NOI is stopped.
  • Such a system may, for example, involve administering the antibiotic tetracycline, to activate gene expression via its effect on the tet repressor/VP16 fusion protein.
  • Another system involves the use of desferrioxamine to induce HRE.
  • tissue-specific promoters will be of assistance in the treatment of disease using the polynucleotides/vectors of the invention.
  • tissue-specific promoters will be of assistance in the treatment of disease using the polynucleotides/vectors of the invention.
  • tissue-specific promoters will be of assistance in the treatment of disease using the polynucleotides/vectors of the invention.
  • several neurological disorders are due to abenant 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.
  • Modified HSCs of the invention are 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 10 6 and of the order of 10 12 cells / dose, preferably at least 10 8 or 10 10 cells per dose.
  • the individual may first be treated to deplete the bone marrow of stem cells or may be treated with one or more cytokines such as G-CSF to increase the mobilisation of stem cells into the peripheral blood or one or more cytokines to enhance repopulation of bone marrow. Combinations of such treatments are also envisaged.
  • the treatments of the invention may also be combined with currently available anti-cancer therapies.
  • the individual suffering from cancer is additionally treated with the corresponding prodrug, administered using an appropriate regimen according to principles known in the art.
  • HSCs are removed from the individual to be treated and are transfected or transduced with the vector in vitro
  • the cells are generally expanded in culture prior to and after introduction of the NOI or NOIs.
  • HSC have the capacity to differentiate into, among other cell types, endothelial cells, myeloid cells, dendritic cells and immune effector cells such as neutrophils, lymphocytes, mononuclear phagocytes and NK cells.
  • tissue culture methods which are known in the art and include exposure to cytokines and/or growth factors for the maintenance of HSCs (Santiago- Schwartz et al, 1992; Charbord et al, 1996; Dao et al, 1997; Piacibello et al, 1997). Agents which induce the differentiation of the HSCs may also be added.
  • HRE elements are known to respond to chemical inducers that mimic hypoxia. Two of these are known, these are cobalt chloride and desferrioxamine (Meliillo et al, 1996; Wang and Semenza 1993b).
  • the products of the invention where HRE is used may also be used to treat a disorder where compounds that mimic hypoxia are administered, such as the chemical activator desferrioxamine or analogous chemicals used to treat neuroblastoma (Blatt, 1994), beta thalassemia (Giardina and Grady, 1995), Alzheimers disease (Crapper et al, 1991), VEGF deficiency (Beenepoot et al, 1996), Erythropoetin deficiency (Wang and Semenza, 1993b) and for enhancement of tumour chemotherapy (Voest et al, 1993).
  • the products of the invention may be adminstered concomitantly, sequentially or separately with the compounds that mimic hypoxia.
  • 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 (Comelisse et al 1996 Pathol Res Pract 192: 684-693) or another relevant gene. It is now recognised that it may be important to identify patients in which a particular product will be safe and effective. This is based on the recognition that genetic variations may contribute to the variable effects of drugs in different individuals.
  • the present invention provides a method of testing an individual's DNA for polymorphisms in drug-metabolizing enzymes and then administering the prodrug activating agent of the present invention if appropriate.
  • Peptides or protein fusions are prepared by making hybrid DNA molecules using standard procedures known to one ordinarily skilled in the art of recombinant DNA technology. For example as described in the literature e.g. Sambrook et al 1989 Molecular Cloning: A Laboratory Approach; Hames and Glover 1985-1997 DNA cloning: a practical approach Volumes I-IV (Second Edition).
  • the hybrid genes are inserted into suitable vectors for transfer to mammalian cells.
  • suitable vectors There are many types of vector available which have been described in detail (e.g. Lever and Goodfellow 1995, Brit. Med. Bull. 51, Number 1, Gene Therapy) and which will be known to ordinary practitioners.
  • a retroviral vector capable of expressing the prodrug activating enzymes described in this and subsequent examples the relevant coding region is inserted into a retroviral vector in which transcripts containing the P450 coding sequences are produced from a strong promoter such as the hCMV-MIE promoter.
  • a suitable plasmid is pHITl l l (Soneoka et al. 1995 Nucl. Acids Res. 23; 628-633) and the required gene is inserted in place of the LacZ gene using standard techniques.
  • the resulting plasmid is then transfected into the FLYRD18 or FLYA13 packaging cell lines (Cosset et al 1995 J. Virol.
  • transfectants are selected for resistance to G418 at 1 mg/ml.
  • G418-resistant packaging cells produce high titres of recombinant retrovirus capable of infecting human cells.
  • the virus preparation is then used to infect human cancer cells and can be injected into tumours in vivo.
  • the prodrug activating enzyme is then expressed from the tumour cells.
  • the MoMLV LTR promoter-enhancer is used for expression of the therapeutic gene in the target cell.
  • the vector can also be modified so that the therapeutic gene is transcribed from an internal promoter-enhancer such as one which is active predominantly in the tumour cells or one which contains a hypoxia regulated element.
  • a suitable HRE-containing enhancer consists of a truncated HSV TK promoter with 3 copies of the mouse PGK HRE (Firth et al. 1994 Proc. Natl. Acad. Sci. 91 : 6496- 6500).
  • the synthetic oligonucleotides described in eg WO95/21927 are inserted between the Nhel and Xbal sites in the 3 'LTR of pHITl l l (Soneoka et al ibid.) to generate a retroviral vector in which gene expression in the target cell is under hypoxia control.
  • An alternative retroviral vector, constructed in the same way, is pKAHRE shown in Figure 6a.
  • Other vector backbones which can be used are shown in Figure 6b.
  • the P450 reductase cDNA is obtained from human placental cDNA by conventional PCR amplification as an EcoRI fragment and ligated into the pBlueScript II vector to form P450R.l
  • the ER anchor domain is removed to produce anchorless P450 reductase (alP450) and a nuclear localisation signal is added.
  • P450 reductase any derivative of P450 reductase (alP450R, see SEQ ID NO: 3 figure 2a for the sequence) that has the SV40NLS substituted for the ER domain is obtained by PCR amplification from the plasmid pP450R.l using the following primers :
  • NLS-alP450 5'-primer SEQ ID NO: 7.
  • alP450 3'-primer SEQ ID NO: 8.
  • the resulting PCR fragment is subcloned into the Smal site of the multiple cloning region of the pCIneo Vector (from Promega).
  • Example 2 The addition of nuclear targeting peptides to the functional domain (FN) of P450R.
  • Figure 7.2 The functional fragment of P450 reductase (FN fragment; (Smith et al 1994, PNAS 91:8710) is shown in Figure 2B.
  • a nuclear targeted FN is constructed as follows :-
  • a PCR fragment of DNA sequence is produced that encodes a fusion protein of :-
  • the PCR fragment is amplified from the pP450R.l (Example 1) using the following primers :
  • FN 5'- primer SEQ ID NO: 12. This contains the Kozak translation initiation sequence lower case, the SV40NLS upper case and the 5 '-region of the FN fragment shown as bold, underlined in the sequence below:
  • FN 3'-Primer SEQ LD NO: 13.
  • the 3'-coding region of the FN fragment is SEQ LD NO: 13.
  • the PCR fragment is subcloned into the Smal site of the pCIneo Vector to produce SV40NLS-FN ( Figure 7.2)
  • Example 3 Construction of a bFGF-alP450R expression vector [ Figure 7.3] Fusions are prepared as described for Example 1.
  • a hybrid protein comprising at the N-terminus of the 18kD isoform of bFGF and at the C-terminus the anchorless P450 reductase (Figure 2A). This is done using two PCR reactions to lift the bFGF sequences from a bFGF-pUC18 plasmid (obtained from R & D systems) to produce a bFGF fragment and to lift the alP450R sequence from p450R.l The two fragments are then digested with Sail and ligated together. The following primers are used:-
  • bFGF 5'-Primer SEQ ID NO: 14. This primes the 5'-coding region of the 18kD isoform with the Kozak leader sequence (underlined).
  • bFGF 3'-Primer SEQ ID NO: 15 . This primes the 3 '-coding region of bFGF with a leading Sail restriction site (shown in upper case)
  • alP450R 5'-Primer SEQ ID NO: 16 . This primes the 5 '-coding region of alP450R with a leading Sail restriction site (shown in upper case)
  • alP450R 3'-Primer SEQ ID NO: 17. This primes the 3'-coding region of alP450R.
  • the two PCR fragments bFGF and alP450R are then digested with Sail, purified and subsequently ligated to each other.
  • the ligated DNA is then subcloned into the Smal site of the mammalian expression vector pCIneo ( Figure 7.3).
  • a flexible linker For example, but not restricted to, the sequence (Gly-Gly-Gly-Gly-Ser) 3 (Somia et al 1993 PNAS 90, 7889). This is a procedure familiar to persons ordinarily skilled in the art of recombinant DNA technology and protein expression.
  • a flexible linker flexlink
  • an alternative set of PCR primers is used to create the following fragments:
  • Primer Sall-flexlink -alP450R SEQ ID NO: 18. Sequences in bold represent rare codons in highly expressed genes (Haas et al 1996 Cun. Biol., 6, 315). Sequences underlined are the 5' regions of alP450R.
  • a cDNA fragment which encodes a fusion protein product of the 18 kD bFGF protein (see Figure 3B) and the FN domain ( Figure 2B) is obtained by PCR amplification from the bFGF/PUC18 plasmid (R&D systems) the FN fragment is obtained from the plasmid pP450R.l using the following primers :
  • bFGF primers For the bFGF 5'- and 3'-primers see Example 3.
  • FN 5'-cPrimer SEQ ID NO: 21. This primes the 5'-coding region of FN with a leading Sail restriction site
  • FN 3'-Primer SEQ ID NO: 13.
  • CTAGCTCCAC ACGTCCAGGG AGTAG (24mer)
  • the two PCR fragments bFGF and FN are then digested with Sail, purified and subsequently ligated to each other.
  • the ligated DNA is then subcloned into the Smal site of the mammalian expression vector pCIneo ( Figure 7.4).
  • Sequences in bold represent rare codons in highly expressed genes (Haas et al 1996 Curr. Biol., 6, 315). Sequences underlined are the 5' regions of FN.
  • SEQ ID NOS: 53 and 54 The Drosophila melanogaster antennapedia protein homeobox peptide (pAntp, Laughon et al 1986 Mol. Cell. Biol. 6:4676)
  • a cDNA fragment which encodes a fusion protein product of pAntp with alP450R is obtained by PCR amplification of pAntp from plasmid ⁇ 903G (Joliot et al 1991 PNAS 88:1864) and amplification of anchorless P450R from the plasmid pP450R.l using the following primers :
  • pAntp 5'-Primer SEQ ID NO: 23. This primes the 5'-coding region of pAntp) with a Kozak leading sequence (underlined).
  • alP450R 5'-Primer SEQ ID NO: 16. This primes the 5'-coding region with a leading Sail restriction site
  • alP450R 3'-Primer Seq ID no 17. This primes the 3 '-coding region
  • the two PCR fragments pAntp and alP450R are then digested with Sail, purified and subsequently ligated to each other.
  • the ligated DNA is then subcloned into the Smal site of the mammalian expression vector pCIneo ( Figure 8.5).
  • a secretion signal can be added to the N-teirninus.
  • a suitable signal is derived from the 5T4 single chain antibody sequence ( Figure 3F).
  • PAntp secretion 5'primer SEQ ID NO: 28. This places a Kozak translation initation site and a secretion signal at the 5' end of the pAntp peptide. Sequences corresponding to pAntp are in upper case, Kozak initiation sequence is underlined.
  • a cDNA fragment which encodes a fusion protein product of the pAntp is obtained by PCR amplification from a pAntp containing plasmid e.g. p903G.
  • the FN fragment (see Figure 2B) is obtained from the plasmid pP450R.l using the following primers :
  • Example 5 For the 5 '-Primer and 3 '-Primer of pAntp, see Example 5 For the 5 ' -Primer and 3 ' -primer of FN, Example 4.
  • the two PCR fragments pAntp and FN are then digested with Sail, purified and subsequently ligated to each other.
  • the ligated DNA is then subcloned into the Smal site of the mammalian expression vector pCIneo ( Figure 7.6).
  • the pAntp secretion 5' primer, SEQ ID NO: 28 can be used.
  • a fusion protein product of VP22 fused to alP450R is obtained by PCR amplification from a VP-22 containing plasmid, pGE109 and by PCR amplification from the plasmid pP450R.l using the following primers:
  • VP22 5' primer SEQ ID NO:29. This primes the 5 '-coding with a Kozak leading sequence (underlined).
  • VP-22 3'-Primer Seq. ED no. 30. This primes the 3'-coding region and contains a Sail restriction site.
  • the two PCR fragments VP-22 and alP450R are then digested with Sail, purified and subsequently ligated to each other.
  • the ligated DNA is then subcloned into the Smal site of the mammalian expression vector pCIneo ( Figure 7.7).
  • a cDNA fragment which encodes a fusion protein product of the VP- 22 is obtained by PCR amplification from pGE109 and the FN fragment (see Figure 2B) is obtained from the plasmid pP450R.l using the following primers :
  • Example 7 For the VP22 5'- and 3'-primers, see Example 7
  • Example 4 For the FN 5'-and 3'-primers, see Example 4
  • the two PCR fragments pAntp and FN are then digested with Sail, purified and subsequently ligated to each other.
  • the ligated DNA is then subcloned into the Smal site of the mammalian expression vector pCIneo [ Figure 7.8].
  • this fusion protein involves the PCR amplification and then ligation of three DNA fragments to form the full insert.
  • the first fragment which encodes a protein product of a single chain variable antibody fragment against the tumour antigen
  • SEQ ID NO: 26 The coding sequence of 5T4 scFv Figure 3F. This is obtained by PCR amplification from a convenient plasmid, pPs-5T4, containing this sequence using the following primers :
  • 5T4scFv 5'-primer SEQ ID NO: 31. This primer provides the translation initiation signal and the secretion signal region from the 5T4scFv sequence ( Figure 3F)
  • 5T4scFv 3'-primer SEQ ID NO: 32. This primes the 3 '-region of the 5T4 scFv fragment with a Hindlll restriction site
  • Fragment 2 PEA fragment.
  • PEA 5'-primer SEQ ID NO: 35. This primes the 5'-coding region with a Hindlll restriction site
  • PEA 3'-primer SEQ ED NO: 36. This primes the 3 '-coding region with a EcoRI restriction site
  • the third cDNA fragment encoding alP450R (see Figure 2A) is obtained by PCR amplification from the plasmid pP450R.l using the following primers :
  • alP450R 5'-primer SEQ ID NO: 37.This primes the 5'-coding region with a EcoRI site
  • alP450R 3'-primers As in Example 3 Seq. ID No 17.
  • the first and the second PCR fragments are digested with Hindlll and then ligated together. Then the ligate and the third PCR fragment are digested with EcoRI and ligated to form the full insert. The full insert is subcloned into the Smal site of the pCIneo expression vector ( Figure 7.9).
  • this fusion protein requires the PCR amplification and subsequent ligation of three DNA fragments to form the full insert.
  • the first fragment encodes a protein product of a single chain variable antibody fragment against the tumour antigen 5T4, as in Example 9A.
  • SEQ ID NO: 26 The coding sequence of 5T4scFv Figure 3F. This is obtained by PCR amplication from a convenient plasmid, pPs-5T4, containing this sequence using the following primers :
  • Fragment 1 5T4 scFv
  • 5T4scFv 5'-primer SEQ ID NO:31. This primer provides the the translation initiation signal and the secretion signal region from the 5T4scFv sequence ( Figure 3F).
  • 5T4scFv 3'-primer SEQ ID NO:32. This primers the 3 '-region with a Hindlll restriction site
  • Fragment 2 alP450R
  • the second cDNA fragment encoding alP450R (see Figure 2A) is obtained by PCR amplification from the plasmid pP450R.1 using the following primers :
  • alP450R 5'-primer SEQ ID NO:38. priming the 5'-coding region with a Hindlll restriction site
  • alP450R 3'-Primer SEQ ID NO:39 priming the 3 '-coding region of alP450R with a EcoRI restriction site
  • the third fragment encoding the membrane translocating sequence (MTS) is obtained by PCR amplification using the following primers :
  • MTS 5'-Primer SEQ ID NO:40 primes the 5'-coding region with a EcoRI restriction site
  • MTS 3'-Primer SEQ ID NO:41 primes the 3 '-coding region
  • the first and the second PCR fragments are digested with Hindlll and then ligated together. Then the ligate and the third PCR fragment are digested with EcoRI and ligated to form the full insert. The full insert is subcloned into the Smal site of the pCIneo expression vector. ( Figure 7.13).
  • Example 10A Construction of a 5T4scFv-PEA-FN expression vector [Figure 7.10]
  • This vector involves the PCR amplification and then ligation of three DNA fragments to form the insert.
  • the first fragment which encodes a protein product of a single chain variable antibody fragment against the tumour antigen 5T4, is obtained as in Example 9A.
  • the second cDNA fragment encoding a protein of the domain II of Pseudomonas aeruginosa exotoxin A (PEA, GenBank accession number KOI 397 and M23348) is obtained as in Example 9 A.
  • PEA 5'-and 3'-primers are as in Example 9A.
  • the third cDNA fragment encoding FN (see Figure 2B) is obtained by PCR amplification from the plasmid pP450R.l using the following primers :
  • FN 5'-primer SEQ ID NO: 32. This primes the 5 '-coding region with an EcoRI restriction site
  • the first and the second PCR fragments are digested with Hindlll and then ligated together.
  • the ligation product and the third PCR fragment are digested with EcoRI and ligated to form the full insert.
  • the full insert is subcloned into the Smal site of the pCIneo expression vector [Figure 7.10].
  • Example 10B Construction of a 5T4scFv-FN-MTS expression vector
  • This vector involves the PCR amplification and then ligation of three DNA fragments to form the insert.
  • the first fragment which encodes a protein product of a a single chain variable antibody fragment against the tumour antigen 5T4, is obtained as in Example 9A.
  • the second fragment encoding FN (see Figure 2B) is obtained by PCR amplification from the plasmid pP450R.l using the follwing primers:
  • FN 5'-Primer SEQ ED NO:43. This Primes the 5 '-encoding region with a Hindlll restriction site.
  • the third fragment encoding MTS is obtained by PCR amplification as in Example 9B.
  • the first and the second PCR fragments are digested with Hindlll and then ligated together. Then the ligate and the third PCR fragment are digested with EcoRI and ligated to form the full insert. The full insert is subcloned into the Smal site of the pCIneo expression vector ( Figure 7.14).
  • SEQ ID NO: 33 The coding region for P4502B6 (Yamano et al 1989 Biochemistry, 28, 7340; GenBank Accession No. JO2864) ( Figure 4). This is subcloned as an Nhel to Xhol fragment into pCI-Neo using the following primers.
  • 2B6 3'primer SEQ ID NO: 35.
  • This primer modifies the C-te ⁇ ninus of the protein to remove the stop codon and replace it with an Xhol site.
  • the PCR fragment is digested with Nhel and Xhol and sub-cloned into pCINeo to produce pCI-Neo containing a truncated P450.
  • a flexi-link FN fragment is constructed using the following primers Flexi-FN 5' primer: SEQ ID NO: 36. This places a Sail restriction site followed by a flexible linker at the N-terminus of the FN fragment. Rare codons in the linker are bold lower case; sequences in FN are underlined. Sail site is upper case.
  • P4502B6 is subcloned into pCINeo as an Nhel/Xhol fragment using the following primers:
  • 2B6 terminus 3' primer SEQ ID NO: 38. This primer retains the authentic translation stop signal
  • Example 13 Construction of a double expression cassette for P450 FN and VP-
  • Combinations of coding regions containing the active entity of P450 and P450R are constructed.
  • One example is the coexpression of VP-FN with P450FN.
  • Example 14A The construction of retroviral vectors expressing modified prodrug activating enzymes.
  • RRVs are produced using a packaging cell line system such as FLYRD18 (Cosset et al).
  • a plasmid vector containing the vector genome to be packaged is transfected into the packaging cell line as described (Cosset FL et al, 1995 J Virol 69 7430-7436) to derive the producer cell line.
  • a suitable plasmid containing vector genome is pHITl l l (Soneoka Y et al 1995 Nucl Acids Res 23 628-633).
  • the required therapeutic gene is inserted in place of the LacZ gene in pHITl 11 using standard molecular biology techniques.
  • Regulatory elements such as HREs and / or enhancer elements may similarly be introduced into the retroviral LTR in pHITl 11 in place of the retroviral enhancer to ensure regulated expression of the therapeutic gene.
  • the plasmid is then co- transfected with a selectable marker gene appropriate for FLYRD18 cells (eg pSV2neo) and transfected cells are selected in 1 mg/ml G418 (Sigma).
  • a selectable marker gene appropriate for FLYRD18 cells eg pSV2neo
  • G418 G418
  • PKAHRE which is an MLV based single transciption unit vector ( Figure 6). Expression of therapeutic genes is controlled by a hypoxia responsive promoter (3xPGK
  • the gene encoding the prodrug activating enzyme is substituted for the nlsLAcZ gene shown.
  • An alternative vector that is particularly suitable for the expression of multiple transcription units is COI ( Figure 6). This is a MLV based vector with the CMV enhancer replacing the MLV enhancer (shaded box). The gene encoding the prodrug activating enzyme is inserted either into the Bam/Sal/Hpa poly linker or the
  • lentiviruses may be used and this is described in more detail in Example 14B.
  • Example 14B Generation of EIAV vector genomes expressing P450
  • peg HRElacZ is described in patent application No. 9901906.9 and is repeated below.
  • pEGASUS4 also pEGASUSl
  • pONY4.0 also pEGASUSl
  • pONY4.1 pONY3.1
  • pHORSE3.1 are described in PCT/GB98/03876 and are also described below.
  • OBhrel Trimer encompassing -307/-290 sequence of murine PGK in the natural orientation (Firth et al, 1995) linked to the SV40 promoter (italicised).
  • This promoter sequence is defined as OBhrel.
  • the resulting plasmid is designated plasmid OB37.
  • hypoxia responsive promoter has been configured into a lentiviral vector, pEGASUS-1 (also pEGASUS-4(+)) and the related vector ⁇ ONY2.1. Both are derived from infectious proviral EIAV clone pSPEIAV19 (Payne et al, 1998; Accession No. U01866). The construction of these plasmids is described below (taken from U.K. patent application no. 9727135.7).
  • Plasmid pONYl was constructed by inserting the EIAV 5' LTR into pBluescript II KS + (Stratagene).
  • the EIAV 5' LTR was amplified by PCR from pSPEIAV19 using pfu polymerase and the following primers - 5' GCATGGACCTGTGGGGTTTTTATGA GG and 3' GCATGAGCTCTGTAGGATCTCGAACAGAC.
  • the amplification product was blunt-ended by 5' overhang fill-in and inserted into pBluescript II KS + cut with BssHII which had been blunt ended by 3' overhang removal using T4 DNA polymerase
  • This construct was called pONYl and the orientation was 5' to 3' in relation to ⁇ -galactosidase of pBluescript II KS + . Sequencing of pONYl revealed no mutations.
  • the env region of pSPEIAV 19 was deleted by removal of the Hind WHind H fragment to generate pSPEIAV19 ⁇ H.
  • the Mlul/Mlul (216/8124) fragment of pSPEIAV 19 ⁇ H was then inserted into pONYl cut with MM to generate a wild type proviral clone (pONY2 ⁇ H) in pBluescript II KS + .
  • a BssHII digest (619/792) of pBluescript II KS + was carried out to obtain the multiple cloning site.
  • pSPCMV was created by inserting pLNCX (Genbank Accession number: M28246) (Pstl/HindlH) into pSP72 (Promega; Genbank Accession No. X65332).
  • the ⁇ -galactosidase gene was inserted from pT_N414 (Cannon et al, 1996) into pSPCMV (Xhol/Sphl) to make pSPlacZ.
  • the 5' end to the ⁇ -galactosidase gene was replaced by the SV40 T-antigen nuclear localization signal from pAD.RSVbgal (Stratford- Pemcaudet et al., 1992) to make pSPnlslacZ (Xhol/Clal).
  • the CMV nuclear localizing and non-nuclear localizing ⁇ -galactosidase from pSPlacZ and pSPnlslacZ was cut out with Pstl and inserted into the Pstl site of ⁇ ONY2.1 in the 5' to 3' orientation of EIAV. These were called pONY2.1nlslacZ and pONY2.11acZ.
  • EIAV-based vector that contains only 759 nt of EIAV sequences (268 nt-675 nt and 7942 nt-8292 nt). Sequences encompassing the EIAV polypurine tract (PPT) and the 3 'LTR were obtained by PCR amplification from pONY2.1 using primers PPTEIAV+ (Y8198): GACTACGACTAGTGTATGTTTAGAAAAACAAGG, and 3'NEGSpeI (Y8199): CTAGGCTACTAGTACTGTAGGATCTCGAACAG.
  • the product was purified, digested with Spel and ligated into pBS II KS + which had been prepared by digestion with Spel and treatment with alkaline phosphatase. Colonies obtained following transformation into E. coli, XL- 1 Blue were screened for the presence of the 3 'LTR in the orientation in which the U5 region of the 3 'LTR was proximal to the Notl site of the pBS II KS + linker. The sequence of the cloned insert was determined and showed that it contained only one change from the EIAV clone pSPEIAV19. This was a 'C insertion between bases 3 and 4 of the R region. The same change was found in the template used in the PCR reaction.
  • the clone was termed pBS.3'LTR.
  • a reporter gene cassette CMV promoter/LacZ
  • the CMV/LacZ cassette was obtained as a Pstl fragment from pONY2.1.
  • the ligation reaction to join the above fragments was transformed into E. coli, XL- lBlue.
  • a number of clones in which the CMV/LacZ insert was orientated so that the LacZ gene was proximal to the 3 'LTR were assessed for activity of the CMV/LacZ cassette by transfection into the cell line 293T using standard procedures.
  • a clone which gave blue cells at 48 hours post-transfection following development with X-gal was selected for further use and termed pBS CMVLacZ.3'LTR.
  • the 5' region of the EIAV vector was constructed in the expression vector pCI-ENeo which is a derivative of pCI-Neo (Promega; Genbank Accession No. U47120) modified by the inclusion of approximately 400 base pairs derived from the 5' end of the full CMV promoter as defined previously. This 400 base pair fragment was obtained by PCR amplifcation using primers VSAT1 (GGGCTATATGAGATCTTGAATAATAAAATG TGT) and VSAT2
  • a fragment of the EIAV genome ninning from the R region to nt 150 of the gag coding region was amplified with primers CMV5 ⁇ IAV2 (GCTAC GCAGAGCTCGTTTAGTGAACCGGGCACTCAGATTCTG: [sequences underlined anneal to the EIAV R region]) and 3'PSI.NEG
  • the PCR product was trimmed with Sad and Xbal and ligated into pCI-Eneo which had been prepared for ligation by digestion with the same enzymes. This manipulation places the start of the EIAV R region at the transcriptional start point of the CMV promoter and transcripts produced thus start at the genuine start position used by EIAV and extend to the 3 '-side of the packaging signal. Clones which appeared to be correct as assessed by restriction analysis were sequenced. A clone termed pCI-Eneo.5'EIAV was selected for further work.
  • the CMVLacZ and 3'LTR cassette in pBS.CMVLacZ.3'LTR was introduced into pCI-Eneo.5'EIAV.
  • pBS.CMVLacZ.3'LTR was digested with Apal, the 3 'overhangs removed with T4 DNA polymerase, then digested with Notl.
  • the fragment containing the CMVLacZ.3'LTR was purified by standard molecular biology techniques.
  • the vector for ligation with this fragment was prepared from pCEEneo.5'EIAV by digestion with Sail, followed by filling-in of the 5'overhangs using T4 DNA polymerase.
  • pEGASUS-1 see which actually shows pEGASUS-4(+), a version of pEGASUS-1 containing an EIAV RRE immediately downstream of the EIAV R-U5-psi region - see below for details).
  • EIAV vector pEGASUS-1 may be made by introduction of additional elements to improve titre.
  • a convenient site for the introduction of such elements is the Sail site which lies between the Xbal to the 3' of the packaging signal and upstream of the CMV/LacZ cassette of pEGASUS-1.
  • the RRE from EIAV can be inserted at this site.
  • the EIAV RRE as defined previously was obtained by PCR amplification as follows. Using pONY2.10LacZ as template two amplifications were performed to obtain the two parts of the EIAV RRE. The 5 '-element was obtained using primers ERRE1 (TTCTGTCGACGAATCCCAGGGGGAATCTCAAC) and ERRE2
  • the second PCR amplification is set up without primers ERRE1 and ERRE4 for the first 10 cycles and then these primers are added to the reaction and a further 10 cycles of amplification carried out.
  • the resulting PCR product and pEGASUS-1 were digested with Sail, ligated and transformed into E.coli XL- 1 Blue.
  • Clones in which the EIAV RRE was in either the positive or negative orientations were selected for further work. These vectors plasmids were called pEGASUS-4(+) (see Figure 20) and pEGASUS-4(-).
  • the CMV promoter in pONY2.1 is excised as an Xbal/Ascl fragment and replaced with an oligonucleotide containing a Mlul/Xbal site. This consequently allows insertion of the Mlul/Xbal fragment isolated from OB37 creating pONY HRE luc/lac. Luciferase coding sequence is removed as an Ncol fragment and the backbone religated creating pONY HRElac. Similarly, lacZ is removed as Xbal/Sall then the backbone religated to create pONY HREluc.
  • the CMV promoter lacZ cassette is excised from the EIAV vector plasmid pEGASUS- 1 with EcoRI and replaced with a synthetic oligonucleotide containing a Sad and a Bsu36 site. This allows the cloning of the HRE luc/HRE lac cassette from pONY HRE luc and pONY HRE lac as Sacl/Bsu36 and SacI/EcoRI fragments respectively.
  • the final vectors are designated pEG-HRE-lacZ and pEG-HRE-luc.
  • pEGHRE vectors may be constructed to express therapeutic genes, such as any of the therapeutic genes listed above, in place of the lacZ or Luc genes. These may be cloned into pEGHRE vectors by excision of the lacZ/luc fragments from pEG-HRE-lacZ or pEG-HRE-luc with Sacl/Bsu36 and SacI/EcoRI, respectively, and ligation of a suitable fragment containing a coding sequence.
  • An example of a therapeutic gene which may be used is the gene encoding anti-angiogenic factor thrombospondin-1 (Genbank Accession No. X04665).
  • an infectious proviral clone pSPEIAV 19 (accession number: U01866), described by Payne et al. (1994, J Gen Virol. 75:425-9).
  • An initial EIAV based vector was constructed by simply deleting part of e «v by removing a Hind Ill/Hind III fragment conesponding to coordinates 5835/6571 according to the numbering system of Payne et al. (ibid.). This fragment was replaced with the puromycin resistance gene under the control of the SV40 early promoter from pTIN500 (Cannon et al 1996 J. Virol. 70:8234-8240) to create pESP.
  • a further vector system was therefore constructed comprising three transcription units to produce the following: 1) vector genome RNA; 2) env and 3) gag-pol.
  • the env and gag-pol transcription units are transcribed from a promoter-enhancer active in the chosen human packaging cell line. In this way, sufficient gag-pol and, most likely tat, are produced to ensure efficient production of transduction-competent vector particles.
  • the vector genome was constructed which has the reporter gene within the pol region of the genome as follows.
  • the plasmid designated pONYl was constructed by inserting the EIAV LTR, amplified by PCR from pSPEIAV 19, into pBluescript II KS+ (Stratagene).
  • the 5' LTR of EIAV clone pSPEIAV 19 was PCR amplified using pfu polymerase with the following primers: 5' GCATGGACCTGTGGGGTTTTTATGAGG
  • the amplicon was blunt ended by 5' overhang fill-in and inserted into pBluescript II KS+ cut with Bss HII which had been blunt ended by 3' overhand removal using T4 DNA polymerase.
  • This construct was called pONYl and the orientation was 5' to 3' in relation to ⁇ -galactosidase of pBluescript II KS+. Sequencing of pONYl revealed no mutations.
  • the env region of pSEIAV19 was deleted at the Hindlll/HindlH sites creating pSPEIAV19DH.
  • Vector genome pSPEIAV19DH was cut with Mlu I (216/8124) and inserted into pONYl Mlu I cut (216) to make pONY2.
  • a Bss HII digest (619/792) of pBluescript II KS+ was carried out to obtain the multiple cloning site. This was blunt ended by 5' overhang fill-in and ligated to pONY2 cut with Bgl II and Nco I (1901/4949) and blunt ended by 5' overhang fill-in. The orientation was 3' to 5' in relation to the EIAV sequence. This was called pONY2.1.
  • pSPCMV was created by inserting pLNCX (Accession number: M28246) (Pst I/Hind III) into pSP72 (Promega). The ⁇ -galactosidase gene was inserted from pTIN414 (Cannon PM et al J. Virol.
  • pAD.RSVbgal J. Clin. Invet. 90:626-630, 1992.
  • pAD.RSVbgal was cut v ⁇ ihXho 1/ Cla I and inserted into Xho 1/ Cla I pSPlacZ to make pSPnlslacZ.
  • the CMV nuclear localizing and non nuclear localizing ⁇ -galactosidase from pSPlacZ and pSPnlslacZ was cut out with Pst I and inserted into the Pst I site of pONY2.1 in the 5' to 3' orientation of EIAV. These were called pONY2.1nlslacZ and pONY2.11acZ.
  • gag-pol gene is expressed from the hCMV-MIE promoter-enhancer.
  • gagpol pSPEIAV 19DH was cut with Mlu I (216/8124) and inserted into pCI-Neo(Promega) Mlu /cut (216) to make ⁇ ONY3.
  • RNA secondary structure prediction (“http://www.ibc.wustl.edu/ ⁇ zuker/ma/") was used to identify possible stem- loop structures within the leader and the 5' end of gag. Based on these predictions four deletions were made within the gag region of pONY2.11acZ. Deletions were made by PCR mutagenesis using standard techniques.
  • pQNY2.111acZ pONY2.11acZ contains 1377nt of gag (deleted from position 1901nt)
  • pONY2.1 llacZ contains 354nt of gag (deleted from position 878nt) PONY3.1
  • pONY3 In pONY3 there is an extended 5' untranslated region before the start of the gagpol coding sequence. It is likely that this unusually long sequence would compromise expression of the gagpol cassette.
  • pONY3 is modified to remove the remaining 5' LTR. This is done by cutting pONY3 with Nar I and Eco RV. The 2.4kb fragment is inserted into pBluescript KS+ (Stratagene) at Cla I and Eco RV sites to make construct pBSpONY3.0. pBSpONY3.0 is cut with_Y7. ⁇ I and Eco RV. The 2.4kb fragment is inserted into pONY3 at Xho I and Eco RV to make pONY3.1.
  • pONY3.1 like pONY3 encodes gag, gagpol, Tat, S2 and Rev. Since the S2 mutation experiments showed that S2 is not required either in the production system or in the EIAV vector genome it is possible to design a gagpol expression constructs without S2. Two such constructs, pHORSE and pHORSE3.1, are produced.
  • PHORSE pHORSE is made by PCR amplification with EGAGP5OUTER EGAGPINNER3 and EGAGP3OUTER/EGAGPINNER5 using ⁇ ONY3 as template DNA.
  • the two PCR products are purified pooled and re-amplified using primers EGAGP5' OUTER EGAGP3' OUTER.
  • This product is inserted into the Xho I and Sal I sites of pSP72 to make pSP72EIAVgagpolO'lap.
  • pONY 3 is cut with Pvu II and Nco I and the 4.3kb fragment is inserted into pSP72EIAVgagpolO'lap cut with Pvu E and Nco I to make pSPEGP.
  • This construct is cut with Xho I and Sal I (4.7kb) and inserted into pCI-Neo at the Xho I and Sal I sites.
  • This construct is called pCIEGP.
  • the RRE is cut out from pEGASUS with Sal I (0.7kb) and inserted into pCIEGP construct at the Sal I site to make pHORSE. When this construct is assayed for protein expression in the presence or absence of pCI-
  • Rev (a construct expressing the EIAV Rev open reading frame, see above) it is found to be Rev dependent as expected. However, protein expression is much lower than from pONY3.1. In addition when used in virus production the titre is found to be 100 fold lower than that from pONY3.1.
  • pHORSE3.1 is made by replacing the 1.5kb Xho VXba I of pHORSE with thel. ⁇ kb Xho VXba I of pONY3.1. Titres obtained with pHORSE3.1 are similar to that of pONY3.1.
  • pQNY4 and QNY4.1 pONY2.1 llacZ contains a deletion in gag such that only 373bp of the gag ORF remains.
  • ⁇ ONY4 was made by replacing the 5' LTR with the CMV LTR from pEGASUS-1.
  • pEGASUS-1 was cut with Bgl WXho I releasing a 3.2kb fragment (containing the CMV LTR) which was inserted into pSP72 cut with Bgl Il/Xho I.
  • This construct was named pSPPEG213. This was cut with Hpa I/Nar l and the 1.3kb fragment (encompassing the CMV LTR) was inserted into pONY2.1 llacZ cut with N ⁇ e I/N ⁇ r I.
  • pO ⁇ Y4.1 contains a deletion (2.1kb) downstream of the lacZ gene (between the Sfu I and Sal I sites) such that tat, S2, env, rev and RRE, are either missing or severely truncated.
  • pONY4.1 was made by cutting it with Sfu I/Sal I, blunt-ended by Klenow polymerase and religated.
  • pONY4G was made by replacing the lacZ gene of pONY4 (Sac II/Kpn I and then blunting with Klenow polymerase) with that of GFP from pEGFP-Nl (Clontech) (Bam Hl/Xba I and then blunting with Klenow polymerase) as a blunt fragment.
  • PCR amplify P450 using (Accession number M29874, also known as CYP2B) primers 5' P450 and 3' P450.
  • the target for the PCR could be liver cDNA or a plasmid containing the P450.
  • P450 PCR fragment is cut with Nco I and the 0.3kb fragment is purified and inserted into pBHRElacZ cut with Nco I.
  • the PCR fragment is cut with Aat Il/Sph I and the 1.3kb fragment inserted into plasmid ⁇ BHREP450del cut with Aat Il/Sph I (3.9kb purified).
  • ⁇ BHREP450 is cut with Not I/sph I releasing the 1.8kb HRE P450 which can be blunt ended with T4 DNA polymerase and inserted into pONY4.0 cut with Pst I and blunt ended to give pONY4HREP450.
  • pBHREP450 is cut with Not I/sph I releasing the 1.8kb HRE P450 which can be blunt ended with T4 DNA polymerase and inserted into pONY4.1 cut with Pst I and blunt ended to give pONY4.1HREP450.
  • PEGASUS4P450 pBHREP450 is cut with Bsm 1/Sph I to give a 1.5kb fragment which is blunt-ended with T4 DNA polymerase and inserted into pEGASUS4 cut with Xho I/Spl I blunt-ended with T4 DNA polymerase to make pEGASUS4P450.
  • pBHREP450 is cut with Bsm I/Sph I to give a 1.5kb fragment which is blunt-ended with T4 DNA polymerase and inserted into pONY4.0 cut with Xho I blunt-ended with T4 DNA polymerase to make pONY4.0P450.
  • PONY4.1P450 pBHREP450 is cut with Bsm I/Sph I to give a 1.5kb fragment which is blunt-ended with
  • the above vector genomes can be used in a three plasmid co-transfection with the EIAV gagpol expression plasmid pONY3.1 and the VSV-G envelope to produce EIAV viral vectors.
  • VSV-G Due to the toxicity of VSV-G it is essential to have some form of regulated expression.
  • a temperature sensitive VSV-G cell line has been described in TE671.
  • the above vector genomes can be used in a three plasmid co-transfection with the EIAV gagpol expression plasmid pONY3.1 and the Rabies-G envelope to produce EIAV viral vectors.
  • Example 15 The use of monocytes/macrophages or stem cells to deliver the enhanced prodrug activating enzymes.
  • Peripheral blood mononuclear cells are isolated from human peripheral blood at laboratory scale by standard techniques procedures (Sandlie and Michaelsen 1996 In Antibody engineering: a practical approach. Ed McCafferty et al Chapter 9) and at large scale by elutriation (eg Ceprate from CellPro).
  • Adherent cells essentially monocytes
  • cells can be allowed to differentiate along the macrophage differentiation pathway by culturing adherent cells for 1-3 weeks.
  • CD14 LPS and LBP receptor (Wright SD et al (1990) Science 249:1431.
  • LPS and LBP receptor Wang SD et al (1990) Science 249:1431.
  • a variety of transfection methods can be used to introduce vectors into monocytes and macrophages, including particle-mediated DNA delivery
  • viral vectors may be used such as defective Adenovirus vectors (Microbix Inc or Quantum Biotechnologies Inc) or retroviral vectors such as those described in figure 6.
  • Stem cells are harvested from peripheral blood after mobilisation with G-CSF and/or cyclophosphamide (Cassel et al 1993 Exp Hematol. 21, 585).
  • G-CSF Amgen
  • Apheresis and enrichment of stem cells is carried out using the CellPro Stem Cell Separator system (Cassel et al op. cit.).
  • the stem cell enriched population is cultured at 10 5 cells/ml in spent medium from RRV producer cells (Example 1), in the presence of 4 g/ml protamine sulphate and 20 ng/ml E -3 (Sandoz), 50 ng/ml IL-6 (Sandoz), 100 ng/ml SCF (Amgen) (Santiago- Schwartz et al 1992, J. Leuk. Biol. 52, 274; Charbord et al 1996, Br. J. Haematol. 94, 449; Dao et al 1997, Blood, 89, 446; Piacibello et al 1997 Blood 89, 2644).
  • cytokines and/or autologous stromal cells prepared as described (Dunbar et al 1996 Hum Gene Ther 7, 231) may also be added. After 24h the cells are centrifuged and resuspended in fresh RRV - containing medium with growth factors and protamine sulphate as above. This is repeated after a further 24h and the cells cultured for up to a further 48h. After this time the cells are trypsinised, washed several times in fresh medium by centrifugation and resuspended in Plasma-Lyte A for re-infusion. The total volume for re-infusion is approximately 25 - 50 ml. Patients are infused over a period of up to two hours. The number of cells infused is at least 10 5 cells and may be up to of the order of 10 12 cells.
  • Cells may also be matured along the myeloid differentiaition pathway prior to re- infusion according to published methods (Haylock et al (1992) Blood 80: 1405-1412).
  • Monocytes, macrophages or stem cells are transfected with an expression vector capable of expressing enhanced prodrug activating enzymes in human cells.
  • Retroviral vectors including lentiviral vectors described above are suitable.
  • the enhanced prodrug activating enzyme is expressed in a vector which utilises the hCMV-MIE promoter-enhancer, pCI (Promega).
  • the hCMV promoter is replaced by a promoter containing at least one HRE.
  • Example 16 The susceptibility of human tumour cells containing modified prodrug activating enzymes to Cyclophosphamide, Tirapazamine and Mitomycin C.
  • Human tumour cell lines (Houlbrook et al 1994, Oncol (Life Sci. Adv.) 13, 69) are obtained from the American Type Culture Collection and grown as monolayers in RPMI 1640 medium supplemented with 2mM glutamine and 10% v/v foetal calf serum.
  • Plasmids are introduced into the human cancer cell lines, in particular the breast cancer MCF-7, MDA468, T47D and NSCLC cell lines using the technique of electroporation as described in Patterson et al ibid. Briefly, cells in exponential growth are harvested with a cell scraper, washed and resuspended in 'cytomix' buffer (Van den Hoff et al 1992, Nucl. Acid Res. 20, 2902. 5 x 10 6 cells are mixed with lOug of linearised plasmid) and subjected to electroporation at 4°C, 960 ⁇ F, 280V, BioRad. Cells are plated at low density and selected for survival in the antibiotic G418.
  • cell lysates are first prepared by liquid nitrogen freezing and thawing cells in buffer comprising lOmM HEPES (pH7.4), ImM EDTA, 0.5mM benzamide, 0.5mM PMSF, lug/ml trypsin inhibitor. Debris is removed by centrifugation at 1600g at 4°C. The resulting supernatent is split into two aliquots. One aliquot stored as a whole cell lysate after adding glycerol to 10%. The other aliquot is centrifuged at 105,000g for 45min at 2°C. The resulting membrane pellet is dried and resuspended in TRIS buffered saline (pH 7.4)
  • the P450 reductase activity is determined spectrophotometrically by measuring the NADPH dependent reduction of cytochrome c.
  • Reactions comprise, 400 ⁇ l of cytochrome c (50uM final concentration), lOOul of lOmM KCN (final concentration ImM) and 10 to 300 ug of protein lysate, which is either the whole cell lysate or the membrane fraction (10-lOOul volume).
  • the reaction is made up to lOO ⁇ l with lOOmM phosphate buffer pH7.6.
  • the reaction is equilibrated to 37°C and initiated by the addition of 20ul of lOmM NADPH (final concentration 200ul).
  • the rate of reduction of cytochrome c is monitored at 550nm for 3min against a blank without NADPH. Initial rates of reaction are calculated and expressed as nmol cytochrome c reduced per min per mg of lysate protein assuming an extinction coefficient of 2 ImM "1 .
  • the concentration of P450 is determined from the CO (carbon monoxide) binding spectra (Omura and Sato, 1964, J. Biol. Chem. 239, 2370)
  • Tirapazamine is synthesised using standard chemistry procedures as described by Seng and Ley, 1972, Angew., Chem., Int. XL 1009 and Adams et al 1984, Br. J. Cancer, 49, 571.
  • Dose response curves are determined using the MTT proliferation assay. This is based upon the ability of viable cells to convert a soluble tetrazolium salt, MTT, into purple formazan crystals (Mossman 1983J. Immunol. Methods. 65, 55). Parallel 96 well plates containing cells seeded at a density of 10 3 per well are incubated for up to 8 days. At daily intervals the plates are assayed for formazan production and cell number is derived from a standard curve of optical density versus cell number generated each day. Values of IC 50 , the concentration of drug required to reduce the optical density by 50% compared to the untreated controls, are used as the measure of cellular sensitivity to a given treatment.
  • Cyclophosphamide is purchased from Sigma Chemical Company. To test for drug sensitivity 4 x 10 4 cells are plated per well of a 6 well culture dish. CP is added 20hrs after seeding. Cells are allowed to grow for up to 7 days. The final viable cell number is determined by the MTT assay as above as above or by staining for viable cells by
  • Mitomycin C is obtained from Sigma Chemical Company. To test for drug sensitivity 5 x 10 3 cells are plated into the wells of a 96 well micro titre plate and after 16hrs they are treated for 3hrs with dilutions of MMC. Cell are allowed to grow for seven days and then cell survival is assayed by the MTT assay as above (Bligh et al 1990 op. cit.)
  • Example 17 The analysis of bystander effects by cocultivation.
  • Cells that have been engineered to express TTS-P450R fusion proteins are mixed with non-engineered cells (control cells) in various ratios.
  • the cell viability after treatment with prodrugs is assessed and the IC 50 is compared with that for homogeneous cultures of engineered cells and control cells.
  • a bystander effect is indicated when percentage cell death exceeds the initial percentage of engineered cells in the mixture.
  • control cells are seeded in a dish and engineered cells are introduced in a COSTAR insert. The drug is added and survival of the control cells is indicative of a diffusible factor arising from the engineered cells. Methods for determining the bystander effect for ep have been described by Chen et al op cit.
  • Example 18 Analysis of the in vivo efficacy of enhanced prodrug activating enzymes.
  • Xenografts of tumour cell lines that have been engineered to express the various enhanced prodrug activating enzymes or combinations are produced in nude mice.
  • the mice are treated with increasing concentrations of the relevant prodrug and tumour growth and cell survival is compared with a control xenograft on the alternate flank.
  • Female homozygous (nuVnu * ) nude athymic Swiss mice 20-30g are used.
  • Tumour cells for example MCF-7 or MDA231, in the exponential growth phase (2 x 10 7 per 0.2ml per injection site) are injected subcutane ously into the flank. Each mouse receives two implants, a control tumour on one flank and a prodrug activating enzyme expressing tumour on the other.
  • mice are pre-implanted with a 17-Beta oestradiol pellet 1 day prior to implanting the tumour cells (Chen et al 1996 op. cit).
  • Drug treatment is initiated when the tumours are approximately 50 to 100mm in size, usually 5 to 6 weeks post implantation. Drugs are administered at an appropriate dose by intra-peritoneal or intra- tumoral injection usually in two administrations separated by 24hrs.
  • tumours are analysed for the expression and action of the prodrug.
  • Tumours are disrupted by standard procedures including mincing, freeze thawing, sonication and treating with coUagenase (500 ⁇ g/ml) depending upon the tumour cell type.
  • Assays for enzyme activity and cell survival are as described above.
  • Example 19 Analysis of the effect of the intra-tumoral delivery of vectors expressing enhanced prodrug activating enzymes.
  • Human tumour xenografts are prepared as described above. Once the xenografts have reach about 50 mm they are injected with a 22 gauge needle with vector preparations. Each tumour is injected at least 5 times to give a distribution of the vector throughout the tumour. The animal is treated with prodrug 48hrs after vector injection and tumours are analysed as described above.
  • Example 20 Macrophages expressing prodrug activating enzyme
  • Primary human macrophages were isolated from peripheral blood by standard methods. They were analysed for the expression of the P450 gene and the P450 reductase gene. Surprisingly they had P450 reductase expression but not P450.
  • Adenoviral vectors were constructed that contain the CMV promoter (Ad.CMV) or the hypoxia responsive HRE promoter (adObHRE).
  • the gene for human cytochrome P4502B6 was inserted into these vectors.
  • P450 expression could be detected in the engineered macrophages.
  • the macrophages were treated with cyclophosphamide and unlike other cells such as tumour cells that have been similarly engineered they were not killed. However, when the P450 engineered macrophages were added to tumour cells or to three dimensional tumour spheroids these were killed in the presence of cyclophosphamide. If the macrophages had been engineered with a marker protein in this case GFP, then the tunour cells all survived. The effect was most dramatic with the Ad.CMV-P450 presumably becuase of the constitutive expression. However, the Ad.HRE-P450 also gave significant killing indicating that one can impose tumour selectivity on the system.
  • Example 21 Killing of human tumour spheroids with macrophages engineered to express human P450
  • the results of this experiment are shown in Figure 9.
  • the top three panels show spheroids that have been treated with engineered macrophages but they have not been treated with cyclophosphamide. They all have a discrete edge and look solid.
  • the bottom panel shows what happens if cyclophosphamide is added.
  • the Ad.CMV- P450 the macrophages the spheroid is completely destroyed and can not be handled for subsequent analysis.
  • Ad.HRE-P450 the spheroid is smaller and more diffuse looking and it is very fragile to handle.
  • Ad.CMV-GFP the spheroid is normal. It is particularly impressive that the tumour target is totally destroyed by Ad.CMV-P450.

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Abstract

L'invention concerne un agent d'activation de promédicaments comprenant: a) un domaine de localisation, et b) un domaine d'activation de promédicaments permettant d'activer un promédicament dans une cellule cible.
PCT/GB1999/000672 1998-03-06 1999-03-05 Activation amelioree de promedicaments Ceased WO1999045126A2 (fr)

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WO2007041546A3 (fr) * 2005-10-03 2007-07-12 Genetix Pharmaceuticals Inc Methode de depletion selective de cellules hypoxiques
WO2007129050A3 (fr) * 2006-05-05 2008-05-02 Univ Montfort Procédés
WO2009120396A3 (fr) * 2008-01-08 2009-12-23 The University Of California Compositions et procédés permettant de réguler l’expression de l’érythropoïétine, d’améliorer une anémie et de stimuler l’érythropoïèse
WO2016195471A1 (fr) * 2015-05-29 2016-12-08 Universidad Nacional Autónoma de México Nanoparticules biocatalytiques cyp-p22 à activité cytochrome p45o pour l'activation de promédicaments
WO2018085586A1 (fr) * 2016-11-02 2018-05-11 David Kiewlich Vecteurs plasmidiques pour l'expression de transgènes d'acide nucléique de grande taille
EP3587582A1 (fr) 2013-10-24 2020-01-01 Adaptimmune Limited Vecteurs d'expression transgénique
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JP5488204B2 (ja) * 2010-05-31 2014-05-14 三菱電機株式会社 サイクロン分離装置及びこれを備えた電気掃除機
CN109535259A (zh) * 2012-12-05 2019-03-29 生控基因疫苗股份有限公司 用作诱发抗原特异性t细胞反应的免疫原性增强剂的融合蛋白
EP3108257B1 (fr) * 2014-02-17 2019-10-30 Société des Produits Nestlé S.A. Procédés et utilisations de mitofusine
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WO2001066579A1 (fr) * 2000-03-07 2001-09-13 Biowindow Gene Development Inc. Shanghai Nouveau polypeptide, proteine humaine 14 de constitution de cytochromes, et polynucleotide codant pour ce polypeptide
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WO2007041546A3 (fr) * 2005-10-03 2007-07-12 Genetix Pharmaceuticals Inc Methode de depletion selective de cellules hypoxiques
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WO2009120396A3 (fr) * 2008-01-08 2009-12-23 The University Of California Compositions et procédés permettant de réguler l’expression de l’érythropoïétine, d’améliorer une anémie et de stimuler l’érythropoïèse
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WO2016195471A1 (fr) * 2015-05-29 2016-12-08 Universidad Nacional Autónoma de México Nanoparticules biocatalytiques cyp-p22 à activité cytochrome p45o pour l'activation de promédicaments
US10480012B2 (en) 2015-05-29 2019-11-19 Universidad Nacional Autonoma De Mexico CYP-P22 biocatalytic nanoparticles with cytochrome P450 activity for prodrug activation
WO2018085586A1 (fr) * 2016-11-02 2018-05-11 David Kiewlich Vecteurs plasmidiques pour l'expression de transgènes d'acide nucléique de grande taille
EP4299736A2 (fr) 2022-04-15 2024-01-03 Vilnius University Hydrolases et leurs utilisations

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WO1999045127A3 (fr) 2000-02-24
WO1999045127A2 (fr) 1999-09-10
EP1068338A2 (fr) 2001-01-17
AU763020B2 (en) 2003-07-10
AU3267099A (en) 1999-09-20
CN1357048A (zh) 2002-07-03
AU3266899A (en) 1999-09-20

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