CHIMERIC TAR ENZYME CONSTRUCTION FOR HIV THERAPY

Abstract: The present invention relates to molecular chimaeras, infective virions, to methods of their construction, to pharmaceutical formulations containing them, to their use in therapy, particularly virus-directed enzyme prodrug therapy particularly in the treatment of HIV, and to the use of agents which can be catalysed by a heterologous enzyme to cytotoxic or cytostatic metabolites, such as purine arabinosides and substituted pyrimidines in virus-directed enzyme prodrug therapy, in a mammalian host (e.g. human).

Description: CHIMERIC TAR ENZYM CONSTRUCTION FOR HIV THERAPY. The present invention relates to molecular chimaeras . infective virions, to methods of their construction, to pharmaceutical formulations containing them, to their use in therapy, particularly virus-directed enzyme prodrug therapy particularly in the treatment of HIV, and to the use of agents which can be catalysed by a heterologous enzyme to cytotoxic or cytostatic metabolites, such as purine arabinosides and substituted pyrimidines in virus-directed enzvme prodrug therapy, in a mammalian host (e.g. human). AIDS is an immunosuppressive or immunodes ruc ive disease that predisposes subjects to fatal opportunistic infections. Characteristically, AIDS is associated with a progressive depletion of T-cells, especially the helper-inducer subset bearing the OKT surface marker. Human immunodeficiency virus (HIV) has been reproducibly isolated from patients with AIDS or with the symptoms that frequently precede AIDS. HIV is cytopathic and appears to preferentially infect and destroy T-cells bearing the OKT marker, and it is now generally recognised that HIV is the etiological agent of AIDS. Since the discovery that HIV is the etiological agent of AIDS, numerous proposals have been made for anti-HIV che otherapeutic agents that may be effective in treating AIDS sufferers. Thus, for example. European Patent Specification No. 196185 describes 3'-azido-3'- deoxythymidine (which has the approved name zidovudine , its pharmaceutically acceptable derivatives and their use in the treatment of human retrovirus infections including AIDS and associated clinical conditions . The numan immunodeficiencv virus vHIV- is composed of a complex genome requiring host-cellular transcriptional factors and viral encoded regulatory peptides for viral gene expression and replication. In addition to the viral rtructural genes (i.e. gag, pol, and env) , HIV also contains at least six regulatory genes (i.e. TAT, rev, vif, nef, upr and upu) which are synthesized from either nascent or spliced message. The TAT gene product has been shown to be a potent transactivator of HIV gene expression and an essential component for the establishment of a productive viral infection. The HIV target sequence for direct or indirect TAT gene product interactions is designated TAR and is located between -17 and +80bp in the 5' long terminal repeat (base pairs numbered relative to the transcription initiation site). More specifically, the TAR sequence most critical for the direct or indirect interactions with the TAT gene product may be located between +1 and +43 in the 5' long terminal repeat. The TAR sequence is present in the HIV proviral DNA as well as in the 5' untranslated portion of all viral mRNAs. In addition to influencing HIV gene expression and replication, recent evidence suggests that the TAT gene product may also influence cellular gene expression and, as such, may also contribute directly to the pathobiology associated with AIDS, such as Kaposi's sarcoma. Transgenic animals containing a transgene composed of the HIV TAT gene developed dermal lesions resembling Kaposi's sarcoma. These data suggest that Kaposi's sarcoma, which occurs in approximately 25% of AIDS patients, may not result from the indirect immunodeficient state, but rather, may be directly and casually associated with expression of HIV TAT. The exact mechanisms of TAT/TAR transactivation mechanisms are unclear. It is clear however, that TAT/TAR interactions can increase HIV gene expression by over 1000-fold, and as such, is essential for a productive viral infection. Gene therapy involves the stable integration of new genes into particular target cells and the expression of those genes, once thev are in place, to alter the phenotype of that particular target cell (for review see Anderson,W.F. Science 226: 401-409, 1984; McCormick, D. Biotechnology 2: 690-693, 1985). Gene therapy may be beneficial for the treatment of genetic disease and involve the replacement of one defective or missing enzyme, such as hypoxanthine-guanine phosphoribosyl transferase in Lesch Nyhan disease; purine nucleoside phosphorylase in severe immunodeficiency disease and adenosine deaminase in severe combined immunodeficiency disease. It has now been found that it is possible to selectively arrest the growth of or kill mammalian cells infected with HIV with chemical agents capable of selective conversion to cytotoxic (causing cell death) or cytostatic (suppressing cell multiplication and growth) metabolites. This is achieved by the construction of a molecular chimaera comprising a transcriptional regulatory sequence (TRS) which is selectively activated in target cells, such as HIV infected T-cells: this controls the expression of a heterologous enzyme. This molecular chimaera may be manipulated via suitable vectors and incorporated into an infective virion. Upon administration of an infective virion containing the molecular chimaera to a mammalian host (eg. human), the enzyme is selectively expressed in the target cell. Administration of compounds which are selectively metabolised by the enzyme into metabolites which are either further metabolised to, or are in fact cytotoxic or cytostatic agents can then be achieved in situ. Molecular chimaera (recombinant molecules comprised of unnatural combinations of genes or sections of genes) , and infective virions (complete viral particles capable of infecting appropriate host cells) are well known in the art of molecular biology and are further described hereinafter. EPO 334 301 describes recombinant retroviruses capable of eliciting selective killing of HIV-infected cells ' .n the presence of nucleoside analogues ACV, AZT or ddC. These compounds mav be modified to cytotoxicity by herpes simplex virus thymidine kinase (HSVTK) encoded by the recombinant retrovirus. Selectively is obtained by placing the HSVTK under control of an HIV tat dependent promoter which will only be expressed in HIV infected cells. A number of enzyme prodrug combinations may be used; providing the enzyme is capable of selectively activating the administered compound either directly or through an intermediate to a cytostatic or cytotoxic metabolite. The choice of compound will depend on the enzyme system used, but must be selectively metabolised by the enzyme either directly or indirectly to a cytotoxic or cytostatic metabolite. The term heterologous enzyme as used herein, refers to an enzyme that is derived from or associated with a species which is different from that which will display the appropriate characteristics of selectivity. The varicella zoster virus (VZV) encodes a specific thymidine kinase (TK) protein. The gene has been cloned, sequenced and characterised (J. Gen. Virol. .67: 1759-1816 (1986)). The VZV thymidine kinase (VZV TK) will, in contrast to the mammalian enzyme, selectively monophosphorylate specific purine arabinosides and substituted pyrimidine compounds. Certain purine and pyrimidine analogues particularly those of Formulae (I) and (II) as hereinafter defined are converted to cytotoxic or cytostatic metabolites in specific mammalian cells which are genetically modified to selectively express VZV thymidine kinase. For example 9-(β-D-arabinofuranosyl)-6-methoxy-9H- purine is converted to 9-(θ-D-arabinofuranosyl adenine triphosphate [Ara ATP] which is cytotoxic. Other enzyme prodrug combinations include the bacterial (for example from Pseudomonas enzyme carboxypepcidase G2 with the prodrug para-N-bis f2-chloroethyl') aminobenzoyi glutamic acid. Cleavage of the glutamic acid moiety from this compound releases a toxic benzoic acid mustard; alkaline phosphatase from, for example, calf intestine, will convert inactive phosphorvlated compounds such as etoposide-phosphate, doxorubicin- phosphate, mitomycin phosphate, to toxic dephosphorylated metabolites. Penicillin-V amidase will convert phenoxyacetamide derivatives of doxorubicin and melphalan to toxic metabolites and cytosine deaminase (for example from E.coli) will convert 5-fluorocytosine to toxic 5-fluorouracil. In mammalian cells, certain genes are ubiquitously expressed. Most genes, however, are expressed in a temporal and/or tissue-specific manner, or are activated in response to extracellular inducers, for example certain genes are actively transcribed only at very precise times in ontogeny in specific cell types or in response to some inducing stimulus. This regulation is mediated in part by the interaction between transcriptional regulatory sequences (which are for example promoter and enhancer regulatory DNA sequences) , and sequence-specific, DNA-binding transcriptional protein factors. It is possible to alter certain mammalian cells, eg. HIV infected T-cells, to selectively express a heterologous enzyme as hereinbefore defined, eg. VZV TK. This is achieved by the construction of molecular chimaeras in expression cassettes. Expression cassettes themselves are well known in the art of molecular biology. Such an expression cassette will contain all essential DNA sequences required for expression of the heterologous enzyme in a mammalian cell. For example, a preferred expression cassette will contain a molecular chimaera containing the coding sequence for VZV TK. an appropriate polyadenylation signal for a mammalian gene (ie. a polyadenylation signal which will function in a mammalian cell) , and enhancers and promoter sequences in the correct orientation. Normally two DNA sequences are required for the complete and efficient transcriptional regulation of genes that encode messenger RNAs in mammalian cells: promoters and enhancers. Promoters are located immediately upstream (5') from the start site of transcription. Promoter sequences are required for accurate and efficient initiation of transcription. Different gene-specific promoters reveal a common pattern of organisation. A typical promoter includes an AT-rich region called a TATA box (which is located approximately 30 base pairs 5' to the transcription initiation start site) and one or more upstream promoter elements (UPE) . The UPEs are a principle target for the interaction with sequence-specific nuclear transcriptional factors. The activity of promoter sequences are modulated by other sequences called enhancers. The enhancer sequence may be a great distance from the promoter in either an upstream (5') or downstream (3') position. Hence, enhancers operate in an orientation- and position-independent manner. However, based on similar structural organisation and function that may be interchanged the absolute distinction between promoters and enhancers is somewhat arbitrary. Enhancers increase the rate of transcription from the promoter sequence. It is predominantly the interaction between sequence-specific transcriptional factors with the UPE and enhancer sequences that enable mammalian cells to achieve tissue-specific gene expression. The presence of these transcriptional protein factors (tissue-specific, trans-activating factors) bound to the UPE and enhancers (cis-acting, regulatory sequences) enable other components of the transcriptional machinery, including RNA polymerase, to initiate transcription with tissue-specific selectivity and accuracy. The selection of the transcriptional regulatory sequence, in particular the promoter and enhancer sequence will depend on the targeted cells. One example, for use in HIV infections, is the transcriptional regulatory sequence for TAR . Other viruses using specific viral transcriptional regulatory sequences include, but are not limited to, human papilloma virus, Epstein Barr virus, and hepatitis B virus. Advantage has been taken of the TA /TAR transactivation interactions to select a suitable transcriptional regulatory sequence. An unnatural molecular chimaera has been created containing the HIV TAR sequence functioning as a conditional transcriptional regulatorv sequence. This TAR sequence regulates the expression of the Varicella zoster virus (VZV) thymidine kinase (TK) gene. Only when the TAR sequence is transcriptionally activated in the presence of an appropriate transactivator (i.e. by direct or indirect interactions with the HIV TAT gene transactivator) will the VZV TK gene be significantly expressed. The VZV TK can selectively metabolise certain nontoxic prodrugs to cytotoxic or cytostatic metabolites, thereby killing or arresting the cells which express VZV TK. Hence, selective expression of VZV TK can be achieved in an HIV infected cell through TAT/TAR interactions. The selective and specific expression of VZV TK will allow the selective production of a cytotoxic or cytostatic compound subsequent to the administration of a nontoxic prodrug. The present invention therefore provides a molecular chimaera comprising a DNA sequence containing the coding sequence of the gene that codes for a heterologous enzyme under the control of the transcriptional regulatory sequence for HIV TAR in an expression cassette. The transcriptional regulatory sequence is thus capable of functioning selectively in a target tissue or cell, for example is capable of transforming an HIV infected cell to selectively express an enzyme for example thymidine kinase. The present invention further provides in a preferred embodiment, a molecular chimaera comprising the transcriptional regulatory sequence for HIV TAR which is selectively activated in mammalian target tissue or cells and is operatively linked to the coding sequence for the gene encoding varicella zoster virus thymidine kinase (VZV TK) . The transcriptional regulatory sequence comprises a promoter and preferably an enhancer. In particular, the present invention provides a molecular chimaera comprising a DNA sequence of the gent coding for the heterologous enzyme, which is preferably VZV TK, including an appropriate polyadenylation sequence, which is linked downstream in a 3' position and in the proper orientation to the transcriptional regulatory sequence for HIV TAR. The DNA sequence encoding a heterologous enzyme is additionally selected from; carboxypeptidase G2; alkaline phosphatase; penicillin -V amidase; and non mammalian cytosine deaminase. The promoter and enhancer sequences preferably are selected from the transcriptional regulatory sequence for HIV TAR. Preferably, the DNA sequence encodes the gene for varicella zoster virus thymidine kinase and is operatively linked to the transcriptional regulatory sequence for HIV TAR. The molecular chimaera of the present invention may be made utilising standard recombinant DNA techniques. Thus the coding sequence and polyadenylation signal of, for example, the VZV thymidine kinase (TK) gene (see Figs. 1A and IB) are placed in the proper 3' orientation to the essential HIV TAR transcriptional regulator elements. Expression of the VZV TK gene in mammalian cells infected by HIV will enable relatively nontoxic arabinosides and pyrimidines as herein defined to be selectively metabolised to cytotoxic and/or cytostatic metabolites. Accordingly, in a further aspect of the present invention, there is provided a method of constructing a molecular chimaera as hereinbefore defined comprising linking a DNA sequence encoding a heterologous enzyme gene, eg. VZV TK to an HIV TAR sequence. In particular the present invention provides a method of constructing a molecular chimaera as herein defined, the method comprising ligating a DNA sequence encoding the coding sequence and polyadenylation signal of the gene for a heterologous enzyme, (eg. VZV TK) to the transcriptional regulatory sequence <,eg. promoter sequence and enhancer sequence) for HIV TAR. The VZV thymidine kinase coding sequence and 3' polyadenylation signal reside in an approximately 1,381 bp Accl/Nde I restriction endonuclease fragment (see Figure IB) . Preferably it is the 1381bp Accl/Nde I fragment containing the VZV TK coding sequence and polyadenylation signal (Figure IB) that is ligated to the promoter and enhancer sequences for HIV TAR, although it will be appreciated that other DNA fragments containing the VZV TK gene could be used. Moreover, the VZV TK polyadenylation signal could be replaced with another suitable polyadenylation signal such as from the SV40 virus or other mammalian genes. These molecular chimaeras can be delivered to the target tissue or cells by a delivery system. For administration to a host (e.g. mammalian or human) it is necessary to provide an efficient in vivo delivery system which stably integrates the molecular chimaera into the cells. Known methods utilise techniques of calcium phosphate transfection, electroporation, microinjection, liposomal transfer, ballistic barrage or retroviral infection. For a review of this subject see Biotechnique Vol.6. No.7. (1988). The technique of retroviral infection of cells to integrate artificial genes employs retroviral shuttle vectors which are known in the art, (Miller A.D. , Buttimore C. , Mol. Cell. Biol 6 2895-2902 (1986). Essentially, retroviral shuttle vectors (retroviruses comprising molecular chimaera as used to deliver and stably integrate the molecular chimaera into the genome of the target cell) are generated using the DNA form of the retrovirus contained in a plasmid. These plasmids also contain sequences necessary for selection and growth in bacteria. Retroviral shuttle vectors are constructed using standard molecular biology techniques well known in the art. Retroviral shuttle vectors have the parental endogenous retroviral genes (eg. gag, pol and env) removed and the DNA sequence of interest inserted, such as the molecular chimaeras which have been described. The vectors also contain appropriate retroviral regulatory sequences for viral encapsidation, proviral insertion into the target genome, message splicing, termination and polyadenylation. Retroviral shuttle vectors have been derived from the Moloney murine leukemia virus (Mo-MLV) but it will be appreciated that other retroviruses can be used such as the closely related Moloney murine sarcoma virus. Other DNA viruses may also prove to be useful as a delivery system. The bovine papilloma virus [BPV] replicates extrachromosomally so that delivery systems based on BPV have the advantage that the delivered gene is maintained in a nonintegrated manner. Thus according to a further aspect of the present invention there is provided a retroviral shuttle vector comprising a molecular chimaera as hereinbefore defined. Preferably the chimaera comprises a transcriptional regulatory sequence for HIV TAR which is selectively activated in target cells and operatively linked to the coding sequence for the gene encoding the heterologous enzyme VZV TK. The chimaera further comprises a DNA sequence of the coding and polyadenylation sequence of the gene coding for VZV TK linked in a 3' position and in the proper orientation to the transcriptional regulatory sequence for HIV TAR to control expression of the VZV TK gene. The DNA sequence encoding VZV TK is operatively linked to a promoter and to a polyadenylation sequence to control expression of the VZV TK gene. The advantages of a retroviral-mediated gene transfer system are the high efficiency of the gene delivery to the targeted tissue or cells, sequence specific integration regarding the viral genome (at the 5' and 3' long terminal repeat (LTR) sequences) and few rearrangements of delivered DNA compared to other DNA delivery systems. Accordingly in a preferred embodiment of the present invention there is provided a retroviral shuttle vector comprising a DNA sequence comprising a 5' viral LTR sequence, a cis acting psi-encapsidation sequence, a molecular chimaera as hereinbefore defined and a 3' viral LTR sequence (Figure 2 ) . In a preferred embodiment, and to help eliminate expression of the molecular chimaera in cells which are not infected with HIV, the molecular chimaera is placed in opposite transcriptional orientation to the 5' retroviral LTR (Figure 2). In addition, a dominant selectable marker gene may also be included which is transcriptionally driven from the 5' LTR sequence. Such a dominant selectable marker gene may be the bacterial neomycin-resistance gene NEO (Aminoglycoside 3' phospho- transferase type II), which confers on eukaroytic cells resistance to the neomycin analogue Geneticin (antibiotic G418 sulphate; registered trademark of GIBCO) (Figure 2). The NEO gene aids in the selection of packaging cells which contain these sequences (see below) . The retroviral vector used in the examples is based on the Moloney murine leukemia virus but it will be appreciated that other vectors may be used. Such vectors containing a NEO gene as a selectable marker have been described, for example, the N2 vector (Eglitin M.A. et al., Science 230 : 1395-1398 (1985)). A theoretical problem associated with retroviral shuttle vectors is the potential of retroviral long terminal repeat (LTR) regulatory sequences transcriptionally activating a cellular oncogene at the site of integration in the host genome. This problem may be diminished by creating SIN vectors (Figure 2). SIN vectors are self-inactivating vectors which contain a deletion comprising the promoter and enhancer regions in the retroviral LTR. The LTR sequences of SIN vectors do not transcriptionally activate 5' or 3' genomic sequences. The transcriptional inactivation of the viral LTR sequences diminishes insertional activation of adjacent target cell DNA sequences and also aids in the selected expression of the delivered molecular chimaera. SIN vectors are created by removal of approximately 299 bp in the 3' viral LTR sequence (Gilboa E. e_t a_l. , Biotechniques « 504-512 (1986)) Thus preferably the retroviral shuttle of the present invention are SIN vectors. Since the parental retroviral gag, pol and βnv genes have been removed from these shuttle vectors, a helper virus system may be utilised to provide the gag, pol and env retroviral gene products in trans to package or encapsidate the retroviral vector into an infective virion. This is accomplished by utilising specialised "packaging" cell lines, which are capable of generating infectious, synthetic virus yet are deficient in the ability to produce any detectable wild-type virus. In this way the artificial synthetic virus contains a chimaera of the present invention packaged into synthetic artificial infectious virions free of wild-type helper virus. This is based on the fact that the helper virus that is stably integrated into the packaging cell contains the viral structural genes, but is lacking the psi-site, a cis acting regulatory sequence which must be contained in the viral genomic RNA molecule for it to be encapsidated into an infectious viral particle. Accordingly in a further aspect of the present invention there is provided an infective virion comprising a retroviral shuttle vector as hereinbefore described, said vector being encapsidated within viral proteins to create an artificial infective, replication-defective retrovirus. Preferably the retroviral shuttle vector comprises a shuttle vector comprising a molecular chimaera having the transcriptional regulatory sequence of HIV TAR. In particular, the retroviral shuttle vector contains an HIV TAR/VZV TK molecular chimaera. In a further aspect of the present invention there is provided a method for producing infective virions of the present invention by delivering the artificial retroviral shuttle vector comprising a molecular chimaera of the invention, as hereinbefore described, into a packaging cell line. The packaging cell line may have stably integrated within it a helper virus lacking a psi site and other regulatory sequence as hereinbefore described, or alternatively the packaging cell line may be engineered so as to contain helper virus structural genes within its genome. In addition to removal of the psi-site, additional alterations can be made to the helper virus LTR regulatory sequences to ensure that the helper virus is not packaged in virions and is blocked at the level of reverse transcription and viral integration. Alternatively, helper virus structural genes (i.e. gag, pol and env) may be individually and independently transferred into the packaging cell line. Since these viral structural genes are separated within the packaging cell's genome, there is little chance of covert recombinations generating wild-type virus. The present invention further provides an infective virion as hereinbefore described for use in therapy, particularly for use in the treatment of HIV infection and pathobiological conditions associated with HIV infection. Selective expression of the heterologous enzyme, in particular VZV thymidine kinase gene is accomplished by utilising tissue-specific, transcriptional regulatory (eg. enhancer and promoter) sequences for HIV TAR target tissues or cells e.g. selectivity may be additionally improved by selective infection of HIV infected cells. The retroviral env gene present in the packaging cell line defines the specificity for host infection. The env gene used in constructing the packaging cell line is modified to generate artificial, infective virions that selectively infect HIV infected cells. As an example, a retroviral env gene introduced into the packaging cell may be modified in such a way that the artificial, infective virion's envelope glycoprotein selectively infect HIV infected cells via the specific receptor mediated binding utilised by HIV. Such modifications of the env gene introduced into the packaging cell may be performed by standard molecular biology techniques well known in the art. The infective virion according to the invention may be formulated by techniques well known in the art and may be presented as a formulation or composition with a pharmaceutically acceptable carrier therefor. Pharmaceutically acceptable carriers, in this instance physiologic aqueous solutions, may comprise a liquid medium suitable for use as vehicles to introduce the infective virion into a host. An example of such a carrier is saline. The infective virion may be a solution or suspension in such a vehicle. Stabilisers and antioxidants and/or other excipients may also be present in such pharmaceutical formulations which may be administered to a mammal by any conventional method eg oral or parenteral routes. In particular, the infective virion may be administered by intra-venous or intra-arterial infusion. Accordingly the invention provides a pharmaceutical formulation or composition comprising an infective virion as hereinbefore described in admixture with a pharmaceutically acceptable carrier. Additionally, the present invention provides methods of making pharmaceutical formulations and compositions as herein described comprising mixing an artificial infective virion, containing a molecular chimaera as hereinbefore described to a cytotoxic or cytostatic metabolite, with a pharmaceutically acceptable carrier. Whilst any suitable compound which can be selectively converted by the enzyme may be utilised, the present invention further provides the use of compounds of Formula (I) or (II) in the manufacture of a medicament for use in the treatment of HIV infected cells capable of expressing VZV thymidine kinase. ό-Substituted purine arabinosides of Formula (I) thereof their salts, esters and physiologically functional equivalents as shown hereinbelow: ^^ (I) wherein M R. is halo, C. ,. alkoxy, halogen-substituted C. alkoxy; an amino group which is mono -or di- substituted by C. alkyl, C. .. alkyl substituted by one or more fluorine atoms, C_ , cycloalkyl, or a nitrogen containing heterocycle containing C, _ carbon atoms and optionally a double bond; and R ? is hydrogen, halo or amino are known as anti VZV and cytomegalovirus agents and their use and synthesis are described in European patent application published under No. 0294114. The following compounds of Formula (I) are preferred compounds to be used in accordance with the present invention; 9-^-D-arabinofuranosyl 6-methylamino-9-H.-purine 9- -D-arabinofuranosy1 6 -dimethylamino- 9-H-pur m ' e . 9-^-D-arabinofuranosyl 6-methoxy-9-H-purine. 9-3-D-arabinofuranosyl 6-ethoxy-9-H-purine. 9-^-D-arabinofuranosyl 6-iodo-9-H-purine. 9-yS-D-arabinofuranosyl 2-amino-6-iodopurine. 9- β -D-arabinofuranosy1 6-pyrrolidino-9-H-purine. 9-?-D-arabinofuranosyl 2-chloro-6-methylaraino-9-H-purine. 9-3-D-arabinofuranosy1 6-cyclopropylamino-9-H-purine. 9-5-D-arabinofuranosyl 6-ethylmethylamino-9-H-purine. 9-3-D-arabinofuranosy1-2-amino-6-methoxy-9-H-purine. 9-β-D-arabinofuranosvl-ό-n-propoxy-9-H-purine. Of the above compounds, 9-3-D-arabinofuranosyl-6-methoxy-9-H-purine is especially preferred. 5-substituted pyrimidine nucleoside compounds of Formula (II) are as shown hereinbelow II ^~ wherein X represents a vinylene or ethynylene group; R represents an 2 oxo or imino group; R represents a hydrogen atom, C. - alkyl, C_ branched or cycloalkyl group e.g. isopropyl or cyclopropyl; R represents a hydrogen atom or an acyl e.g. C- alkanoyl or benzoyl group optionally substituted for example by one or more halogen, alkyl, hydroxy or alkoxy substituents; and R represents a hydrogen atom or a hydroxy group. 3 It will be appreciated that when R is not an acyl group, a compound of Formulae (I) or (II) may exist in its tautomeric form. The following compounds of Formula (II) are preferred compounds to be used in accordance with the present invention: 2'-Deoxy-5-(l-propynyl)uridine. 2' -Deoxy-5-ethynylcytidine . 3-N-Benzoyl-2 ' -deoxy- 5-ethvnyIuridine . I- (ϋ3-D-Arabinofuranosyl ) - 5-ethynyluracil . 2 ' -Deoxy-5- (l-propynyl)cytidine . 1- <"3-D-Arabinofuranosyl) -5 -propynylcytosine . 3-N-Benzoyl-2' -deoxy-5-propynyluridine . 1-(^-D-Arabinofuranosyl)-5-propynyluracil. 1- (β-D-Arabinofuranosy1)-5-ethynylcytosine. 1- (?-D-Arabinofuranosyl)-3-N-benzoyl-5-propynluracil. 1-(?-D-Arabinofuranosy1)-3-N-benzoyl-5-ethynyluracil. 3-N-Benzoyl-2' -deoxy-5-vinyluridine. . β-Q-Arabinofuranosyl)-3-N-benzoyl-5-vinyluracil. A particularly preferred compound of Formula (II) is 1-(3-D-Arabino- furanosyl) -5-propynyluracil. These compounds, and methods of their synthesis have been disclosed in European Patent application published under No. 0272065 as having anti VZV activity. The above-mentioned pyrimidine nucleosides and purine arabinosides also include the pharmaceutically acceptable derivatives of such compounds, ie. any pharmaceutically acceptable salt, ester, or salt of such ester, or any other compound which, upon administration to a human subject, is capable of providing (directly or indirectly) the active metabolite or residue thereof. Preferably the compound is orally active. The amounts and precise regime in treating a mammal, will of course be the responsibility of the attendant physician, and will depend on a number of factors including the type and severity of the condition to be treated. Prodrug treatment - Subsequent to infection with the infective virion, compounds according to the invention which are described by Formulae (I) and (II) are administered, that specifically require VZV TK activitv for the critical phosphorylation step in anabolism to generate cytotoxic or cvtostatic metabolites. The prodrug compounds, which are subsequently converted to cytotoxic or cytostatic metabolites in the target cells, are preferably purine arabinosides or pyrimidine nucleosides. Most preferably 9-?-D-arabinofuranosyl-6- methoxy-9H-purine and 1-(3-D-arabinofuranosyl)-5-propynyluracil. The above mentioned prodrug compounds are administered to the host (e.g., mammal or human) between six hours and ten days, preferably between one and five days, after administration of the infective virion. The dose of compound as described by Formulae (I) or (II) to be given will advantageously be in the range 0.1 to 250 mg per kilogram body weight of recipient per day, preferably 0.1 to lOOmg per kilogram bodyweight. More preferably the dose is 1 to 40mg per kilogram bodyweight most preferably 15-40mg per kilogram bodyweight. The invention also provides a method of treating a host (e.g., mammal or human) in need of anti HIV treatment which comprises administering to the host, a molecular chimaera, as described herein, which is capable of being selectively activated in the HIV infected cells of the host to express an enzyme, and subsequently administering an agent which is converted in the cells by the enzyme to an agent which is cytotoxic or cytostatic to the cells. The invention further provides a method of treating a host in need of anti-HIV treatment comprising administering to the host an infective virion as described hereinbefore. Namely, the infective virion encapsidating a retroviral shuttle vector comprising a molecular chimaera, the chimaera comprising a transcriptional regulatory sequence for HIV TAR which is selectively activated in the cells of the host and operatively linked to a gene encoding a heterologous enzyme: in an amount sufficient to transform the cells so as to express the enzyme, and subsequently administering to the host an amount of a compound which is selectively metabolised in the cells by the enzvme to a cvtotoxic or cvtostatic metabolite. FIGURE LEGENDS Figure 1A Diagram of Varicella Zoster Thymidine Kinase Gene. Figure IB VZV TK gene - 1 sequence. Figure 2 Proviral form of retrovirus containing HIV TAR/VZV TK molecular chimaera Figure 3 ml3mpl9*LTR. Figure 4 pCR73 and ml3mpl9*LTR Figure 5 N2LTRTK. The following examples serve to illustrate the present invention but should not be construed as a limitation thereof: Example 1 Construction of the HIV-LTR driven VZV-TK pUCHIV (M. Davis e_t £l PNAS 84,1978.p 8642-8646.) was digested with Hindlll and BamHI. The HIV LTR was isolated by agarose gel electrophoresis, electoeluted and subcloned into ml3mpl9 (Yanisch-Perron, C. et al 1985 Gene 33:103) (Available from New England Biolabs., Beverly, Massachusetts). Mutations were introduced by oligonucleotide-directed mutagenesis to create an Xhol restriction endonuclease site at the 5' end of the HIV-LTR. The plasmid was designated ml3mpl9*LTR. Mutations were confirmed by sequencing. See Fig 3. pUCHIV was deposited at the American Type Culture Collection, Rockville MD USA (ATCC) on 27th February 1991 under the Budapest Treaty with Accession No. 40982 ml3mpl9*LTR was deposited at the American Type Culture Collection, Rockville MD USA (ATCC) on 27th February 1991 under the Budapest Treaty with Accession No. 40984 pCR73 (deposited at ATCC under Number 68077) was digested with BAMHI and Kpnl to enable the isolation of the VZV-TK containing fragment. The VZV-TK DNA fragment was subcloned into the BamHI and Kpnl digested ml3mpl9*LTR plasmid. Plasmids containing the VZV-TK (mpl9*LTR/TK) were sequenced to ensure the subcloned DNA integrity. See Fig. 4. mP19*LTR/TK was deposited at the American Type Culture Collection, Rockville MD USA (ATCC) on 27th February 1991 under the Budapest Treaty with Accession No. 40983 The HIV-LTR/VZV-TK containing DNA was isolated as an Xhol fragment and subcloned into N2(XM5). Plasmids were screened for orientation. Plasmids (denoted N2LTR/TK) were chosen which would allow only HIV-LTR driven VZV-TK expression in TAT expression cell lines. See Fig. 5. N2LTR/TK was deposited at the American Type Culture Collection, Rockville MD USA (ATCC) on 27th February 1991 under the Budapest Treaty with Accession No. 40985

Claims: CLAIMS 1. A molecular chimaera comprising a transcriptional regulatory sequence for HIV TAR capable of being selectively activated in a mammalian target cell, a DNA sequence operatively linked to the transcriptional regulatory sequence and encoding a heterologous enzyme, the enzyme being capable of catalysing the production of an agent toxic to the target cell. 2. A chimaera as claimed in Claim 1 wherein the transcriptional regulatory sequence comprises a promoter. 3. A chimaera as claimed in Claims 1 or 2 wherein the transcriptional regulatory sequence additionally comprises an enhancer sequence. 4. A chimaera as claimed in Claims 1, 2 or 3 additionally comprising a polyadenylation signal downstream of the DNA sequence encoding the heterologous enzyme. 5. A chimaera as claimed in any one of Claims 1 to 4 wherein the encoded enzyme is varicella zoster virus thymidine kinase. 6. A chimaera as claimed in any one of Claims 1 to 4 wherein the encoded enzyme is selected from; carboxypeptidase G2, alkaline phosphatase, penicillin-V amidase and non-mammalian cytosine deaminase. 7. A retroviral shuttle vector containing a molecular chimaera as claimed in any one of Claims 1 to 6. 8. A vector as claimed in Claim 7 comprising a 5' viral long terminal repeat (LTR) sequence, a cis-acting psi encapsidation sequence, a molecular chimaera of any one of Claims 1 to 6 and a 3' viral LTR sequence. 9. A vector as claimed in Claim 8 wherein the molecular chimaera is oriented in the opposite direction to the 5' retroviral LTR. 10. A vector as claimed in Claim 8 or 9 additionally comprising a DNA sequence encoding a dominant selectable marker operatively linked to the 5' LTR sequence. 11. A vector as claimed in Claim 10 wherein the selectable marker sequence is a NEO gene. 12. A vector as claimed in any one Claims 7 to 11 being a self inactivating (SIN) vector. 13. An infective virion encapsidating a vector as claimed in any of Claims 7 to 12. 14. An infective virion as claimed in Claim 13 engineered so as to selectively infect a target cell. 15. An infective virion as claimed in either of Claims 13 or 14 which is replication-defective. 16. An infective virion as claimed in any of Claims 13 to 15 wherein the env gene of the virion is modified so as to facilitate cell specific viral uptake. 17. An infective virion as claimed in Claim 16 wherein the env gene is modified so as to preferentially infect cells which are HIV infected. 18. A molecular chimaera as claimed in any one of Claims 1 to 10 for use in medical therapy. 19. An infective virion as claimed in any one of Claims 13 to 17 for use in medical therap2>'. 20. Use of an infective virion as claimed in any one of Claims 13 to 17 in the manufacture of a medicament for use in the treatment of HIV infection and pathobiological conditions associated with HIV infection. 21. Use of a compound in the manufacture of a medicament for treating HIV infected cells capable of expressing a heterologous enzyme, said compound being selectively catalysed by the enzyme either directly or through an intermediate to a cytotoxic or cytostatic metabolite. 22. Use of a compound in the manufacture of a medicament for treating HIV infected cells which are capable of expressing VZV thymidine kinase wherein the compound is of formula (I): (I) **c wherein R. is halo, C alkoxy, halogen-substituted C alkoxy; an amino group which is mono- or di- substituted by C. alkyl, C . alkyl substituted bv one or more fluorine atoms, C, , cvcloalkvl, or a nitrogen containing heterocycle containing C carbon atoms and optionally a double bond; and R. is hydrogen, halo, amino or a salt or physiologically functional derivative thereof, or of formula (II) : wherein X represents a vinylene or ethynylene group: R represents an oxo 2 or imino group; R represents a hydrogen atom, C. „ alkyl, C, , branched or cycloalkyl group e.g. isopropyl or cyclopropyl; R represents a hydrogen atom or an acyl e.g. C. alkanoyl or benzoyl group optionally substituted for example by one or more 4 halogen, alkyl, hydroxy or alkoxy substituents; and R represents a hydrogen atom or hydroxy group. 23. Use of a compound or mixture of compounds according to Claim 22 selected from the following: 9-^-D-arabinofuranosyl-6-methylamino-9-H-purine. 9- -D-arabinofuranosyl-6-dimethylamino-9-H-purine . 9- 3-D-arabinofuranosyl-6-methoxy-9-H-purine . 9-jS-D-arabinofuranosyl-6-ethoxv-9 -H-purine . 9- ?-D-arabinofuranosyl-6 -ϊodo-9-H-purlne . -β-D-arabinofuranosyl-2-amino-6-iodopurine. 9 -β-D -arabinofuranosy1-6-pyrrolidino-9-H-purine. 9- -D-arabinofuranosyl-2-chloro-6-methylamino-9-H-purine. 9-#-D-arabinofuranosyl-6-cyclopropylamino-9-H-purine. 9- β-D-arabinofuranosy1-6-ethylmethylamino-9-H-purine. 9 -β-D-arabinofuranosyl-2-amino-6-methoxy-9-H-purine. 9-0-D-arabinofuranosyl-6-n-propoxy-9-H-purine. 2' -Deoxy-5-(l-propynyl)uridine. 2' -Deoxy-5-ethynylcytidine. 3-N-Benzoyl-2' -deoxy-5-ethynyluridine. 1-(3-D-Arabinofuranosyl)-5-ethynyluracil. 2' -Deoxy-5-(l-proρynyl)cytidine. 1- (^-D-Arabinofuranosyl)-5-propynylcytosine. 3-N-Benzoyl-2' -deoxy-5-propynyluridine. 1-(^-D-Arabinofuranosyl)-5-propynyluracil. 1-(^-D-Arabinofuranosyl)-5-ethynylcytosine. 1-(^-D-Arabinofuranosyl)-3-N-benzoyl-5-propynluracil. 1-(^-D-Arabinofuranosyl)-3-N-benzoyl-5-ethynyluracil. 3-N-Benzoyl-2' -deoxy-5-vinyluridine. 1-(9-D-Arabinofuranosyl)-3-N-benzoyl-5-vinyluracil. or salts or physiologically functional derivatives thereof. 24. Use of 9-(/3-D-arabinofuranosyl-6-methoxy-9-H-purine or a salt, or physiologically functional derivative thereof according to Claim 22. 25. Use of an infective virion as claimed in Claims 13 to 17 and a compound which can be selectively catalysed by a heterologous enzyme to cytostatic or cytotoxic metabolite for concomitent use in therapy. 26. A process for the production of a molecular chimaera comprising a transcriptional regulatory sequence for HIV TAR capable of being selectively activated in a mammalian target cell, a DNA sequence operatively linked to the transcriptional regulatory sequence and encoding a heterologous enzyme, the enzyme being capable of catalysing the production of an agent toxic to the target cell.

Inventors: HUBER BRIAN, MARTIN JEAN LOUISE, RICHARDS CYNTHIA ANN, HUBER, BRIAN, MARTIN, JEAN, LOUISE, RICHARDS, CYNTHIA, ANN
Applicants: WELLCOME FOUND, THE WELLCOME FOUNDATION LIMITED
Application year: 1992
Main classification: C12N15
IPC codes: A61K31, A61K38, A61K48, A61P31, C07H19, C07K14, C12N9