EP1080100A1 - Cytosine deaminase gene - Google Patents

Abstract

The present invention relates to a cytosine deaminase (CD) gene optimal for gene or viral directed enzyme prodrug therapy (GDEPT or VDEPT). The invention further relates to recombinant molecules comprising the CD gene and to host cells comprising same. The invention also relates to methods of effecting GDEPT or VDEPT using the CD gene.

Description

CYTOSINE DEAMINASE GENE

Technical Field

The present invention claims priority to application GB9810752.7 filed 19 May 1998 in Great Britain and relates to a cytosine deaminase (CD) gene. In particular, but not exclusively, the CD gene is useful for gene or viral directed enzyme prodrug therapy (GDEPT or VDEPT). The invention further relates to recombinant molecules comprising the CD gene and to host cells or infective virions comprising same. The invention also relates to methods of effecting GDEPT or VDEPT using the CD gene.

Background

Microorganisms have many distinct metabolic pathways which are absent in mammalian cells. It is through these pathways that relatively nontoxic prodrugs are metabolized to highly toxic forms. Drugs like acyclovir, 5-fluorocytosine (5-FC) and ganciclovir, developed for the treatment of infections, often target these unique enzyme pathways. Transfer of genes encoding these distinctive enzymes, sometimes called suicide genes, to mammalian cells confers upon the resulting, genetically altered cells novel chemosensitivity to prodrugs.

Cytosine deaminase (CD; EC 3.5.4.1) catalyzes the deamination of cytosine to uracil (O'Donovan et al, Bact. Rev.

34:278 (1970)). Mammalian cells do not ordinarily produce this enzyme (Nishiyama et al, Cancer Res. 45:1753 (1985)), which plays an important role in microbial metabolism, and has been isolated from several different microorganisms. Many bacteria and fungi that express the CD gene convert 5-fluorocytosine (5-FC) to

5-fluorouracil (5-FU), a highly toxic metabolite that is lethal to the cell. Since normal mammalian cells do not express significant amounts of CD, they are incapable of deaminating 5-FC to the toxic metabolite 5-FU, and therefore 5-FC is nontoxic to mammalial cells at concentrations that result in strong antimicrobial activity. However, 5-FU has potent cytotoxic effects on mammalian cells and is widely used as an anticancer agent. Therefore, mammalian cells engineered to express CD should convert 5-FC to 5-FU and be selectively sensitive to 5-FC, compared to native, unmodified cells.

The CD gene has recently been isolated and cloned from Escherichia coli (Austin et al, Mol. Pharmacol. 43:380 (1992); Anderson et al, Arch. Microbiol. 152:115 (1989)) and from Saccharomyces cerevisiae or baker's yeast (EP Application No.

90306131.5). Various investigators have shown that transfer of the E. coli gene to mammalian cells renders those cells selectively sensitive to 5-FC in vitro (Austin et al Mol. Pharmacol. 43:380 (1992); Mullen et al, Proc. Natl. Acad. Sci. USA 89:33 (1992)). It has also been demonstrated that tumor cells which have been engineered with retroviral vectors to express the E. coli CD gene can be eliminated in vivo by systemic treatment of animals with 5- FC (Huber et al, Cancer Res. 53:4619 (1993); Mullen et al, Cancer Res. 54:1503 (1994); Huber et al, Proc. Natl. Acad. Sci. USA 91 :8302 (1994)). A replication deficient adenovirus vector

(Hirschowitz et al, Human Gene Ther. 6:1055 (1995)) and cationic liposomes (Davis et al, Proc. AACR Abstract No. 2355, p345 (1996)) have also been used to transfer the E. coli CD gene to human colon carcinoma cells and large cell lung carcinoma tumors in mice, respectively. Expression of the gene in these tumor cells conferred sensitivity to 5-FC.

Although substantial antitumor effects were observed in these studies, the effects were only observed at the highest 5-FC dose tested (500 mg/kg body weight for about 15 days), a dose level which exceeds the solubility limit of 5-FC and requires administration as suspension (Huber et al, Cancer Res. 53:4619 (1993)). A corresponding dose of 5-FC in humans also showed some myelosuppressive effects (Bennett, J. E. 1990 in Goodman's and Gilman's The Pharmacological Basis of Therapeutics, eds. Gilman A.G., Rail, T., Nies, A.S. & Taylor, P., Pergamon, New York, 8th Edition, pp 1165-1181 ). A CD gene that more efficiently expresses an enzyme in tumor cells would lower the amount of 5- FC needed to achieve these antitumor effects, and thus would improve the therapeutic index of 5-FC.

It is an object of the present invention to provide a CD gene which is efficiently expressed in mammalian cells. Other objects of the present invention will become apparent from the following description thereof. Throughout this specification and the appended claims, unless the context requires otherwise, the words comprise and include or variations such as comprising, comprises, including, includes, etc., are to be construed inclusively, that is, use of these words will imply the possible inclusion of integers or elements not specifically recited.

Summary

According to one embodiment of the present invention there is provided a nucleic acid sequence encoding cytosine deaminase

(CD), wherein said nucleic acid sequence has a guanine : cystocine (G : C) content higher than that of a naturally occurring

CD encoding nucleic acid sequence.

According to another embodiment of the invention the nucleic acid sequence comprises codons optimal for expression in a mammalian host cell.

According to another embodiment of the invention the nucleic acid sequence is between about 300 and about 500 times as efficient as an E. coli CD gene in converting 5-FC to 5-FU, in mammalian cells.

According to another embodiment of the present invention there is provided a nucleic acid sequence which is the same as or has substantial identity to the sequence of SEQ ID NO: 4. According to another embodiment of the invention there is provided a pharmaceutical composition comprising the nucleic acid sequence and a pharmaceutically acceptable carrier.

According to a still further embodiment of the invention there is provided a liposome comprising the nucleic acid as referred to above.

The invention further relates to recombinant molecules

(including expression cassettes) that comprise this gene and host cells (including human tumor cells) transformed therewith. The invention also relates to methods of using the CD gene of the invention for GDEPT or VDEPT.

Brief Description of the Drawings

Figure 1. Yeast CD (YCD) DNA sequence (EPA

No. 90306131.5) (SEQ ID NO:1 ). Nucleotides in boxes were changed to generate the CD gene of the invention. The yeast CD sequence was isolated from commercially prepared S. cerevisiae DNA (Clontech Laboratories Inc.). Using this DNA as template, oligonucleotide primers YCD#1 (δ'-AAACTGCAGAAGCTTGCCA-

CCATGGTGACAGGGGGAATGGCAA-3') (SEQ ID NO:2) and YCD#2 (5'-GGGGAATTCTCTAGACTACTCACCAATATCTTCAAA- CCAA-3') (SEQ ID NO:3) that bound at the 5'- and 3'-end of the yeast CD sequence, respectively, and Vent DNA polymerase, a 513 base pair (bp) fragment was amplified. The 513bp fragment was purified through an agarose gel and restricted with Hind III at the 5'-end and Xba 1 at the 3'-end. These enzyme sites were engineered into the primers used for amplification. The yeast CD sequence was subcloned into an appropriate vector (pRc/ CMV) cut with the same two restriction enzymes.

Figure 2. DNA sequence of a chemically synthesized CD (CS-CD) of the invention (SEQ ID NO:4). Figure 3. Expression of CD genes in H460 cells as a function of DNA concentration. Expression in mammalian cells was performed by carrying out transient transfections into human lung large cell carcinoma cells (H460). Transfections were carried out by liposome-mediated DNA delivery using lipofectamine (Life

Technologies, Inc., Gaithersburg, MD, USA) as per manufacturer's instructions. Briefly, 5x10⁵ - 1x10⁶ cells were seeded in a 60 mm tissue culture plate. After transfections using either pCMV/E. coli CD, pCMV/YCD or pCMV/CS-CD (2 μg DNA), cells extracts were prepared by scrapping off the cells in a solution containing 0.1%

Triton-X 100 and 5FM PMSF in 1x PBS. The whole cell extracts were assayed for the enzyme activity or stored at -80°C. The enzyme activity was measured in a reaction mix containing phosphate buffered saline, 4mM 5-FC or cytosine, O.δμCi [³H]5-FC or [³H]cytosine (Moravek Biochemical Inc., CA, USA) and an appropriate amount of enzyme. Enzyme reactions were carried out at 37°C for different times and stopped by the addition of 24 volumes of 1 M acetic acid. [³H]5-FU or [³H]uracil was separated using a Bond-elute column (Varian) and estimated. One unit of activity was defined as the amount of enzyme that generated

1 μmol of [³H]5-FU or [³H]uracil per minute at 37°C. (Specific Activity (μmol/min/mg protein) = 0.02 at 1 μg E. coli CD; 0.05 at 2 μg E. coli CD, 0.13 at 4 μg E. coli CD; 0.14 at 6 μg E. coli CD; 0 at 1 μg yeast CD; 0.02 at 2 μg yeast CD; 0.11 at 4 μg yeast CD; 0.11 at 6 μg yeast CD; 2.19 at 1 μg CS-CD; 15.71 at 2 μg CS-CD;

42.39 at 4 μg CS-CD; and 48.87 at 6 μg CS-CD).

Figure 4. Expression of CD gene of the invention in human lung adenocarcinoma cells (A549) as a function of DNA concentration. (Specific activity (μmol/min/mg protein) = 0.011 at 0.5 μg E. coli CD; 0.043 at 1 μg E. coli CD; 0.14 at 1.5 μg E. coli

CD; 0.203 at 2 μg E. coli CD, 0.35 at 3 μg E. coli CD; 0.83 at 4 μg E coli CD; 0.0002 at 0.5 μg yeast CD; 0.002 at 1.0 μg yeast CD; 0.033 at 1.5 μg yeast CD; 0.064 at 2 μg yeast CD; 0.347 at 3 μg yeast CD; 0.798 at 4 μg yeast CD; 0.586 at 0.5 μg CS-CD; 4.63 at 1 μg CS-CD; 13.08 at 1.5 μg CS-CD; 75.99 at 2 μg CS-CD; 98.195 at 3 μg CS-CD; 132.76 at 4 μg CS-CD.

Figures 5A-5D. Chimeras of yeast CD and CS-CD. Fig. 5A shows the sequence of YCDM #A (SEQ ID NO:5). The sequence in bold corresponds to the yeast CD sequence. The rest of the sequence corresponds to the CS-CD sequence. Fig. 5B shows the sequence of YCDM #B (SEQ ID NO:6). The sequence in bold corresponds to the yeast CD sequence. The rest of the sequence corresponds to the CS-CD sequence. Fig. 5C shows the sequence of YCDM #C (SEQ ID NO:7). The sequence in bold corresponds to the yeast CD sequence. The rest of the sequence corresponds to the CS-CD sequence. Fig. 5D shows the sequence YCDM #D (SEQ ID NO:8). The sequence in bold corresponds to the yeast CD sequence. The rest of the sequence corresponds to the CS-CD sequence. The underlined sequence represents an altered sequence resulting in a change in the reading frame and a premature stop that shortens the protein by six amino acids.

Figures 6A-6D. Mutants of the protein encoded by CS-CD.

Fig. 6A shows the nucleic acid (SEQ ID NO:9) and protein (SEQ ID NO: 10) sequences of YCDM #1. The sequence in bold represents a change in the amino acid sequence from lie to Leu. Fig. 6B shows the nucleic acid (SEQ ID NO:11 ) and protein (SEQ ID NO:12) sequences of YCDM #2. The sequence in bold represents a change in the amino acid sequence from Thr to Ser. Fig. 6C shows the nucleic acid (SEQ ID NO:13) and protein (SEQ ID NO:14) sequences of YCDM #3. The sequence in bold represents a change in the amino acid sequence from Arg to Lys. Fig. 6D shows the nucleic acid (SEQ ID NO:15) and amino acid (SEQ ID

NO: 16) sequences of YCDM #4. The sequence in bold represents a change in the amino acid sequence from Lys to Arg. Detailed Description of the Invention

Surprisingly, the present inventors have found that by altering the nucleic acid sequence of yeast CD by conservative mutation, to increase the level of GC content, the level of CD expression in mammalian cells is also markedly increased. In this regard "conservative mutation" is intended to mean that alterations to codons within the nucleotide sequence do not change, or at least do not substantially change, the amino acid sequence encoded thereby, the result being that an active CD enzyme is still produced.

The coding sequence of yeast CD (consisting of 477 nucleotides coding for a protein of approximately 158 amino acids) is shown in Fig. 1. A comparison of the DNA sequences of the E. coli CD gene

(Austin et al, Mol. Pharmacol. 43:380 (1992)) and the CD gene from baker's yeast (EP Application No. 90306131.5) revealed that the GC content was 53.3 % for E. coli CD gene vs 44.4% for yeast CD gene. In the CD encoding sequence of the present invention, codons that are preferred in a mammalian (especially human) host (see codon usage table in Wada et al, Nucleic Acid Res. 19:1981 (1991 )) and that provide a higher GC content, replace the non- preferred codons of the yeast CD encoding sequence. Such codons are said to be "optimal" for expression in a mammalian host cell, in that by utilising these codons an improved level of CD expression is obtained in mammalian cells, relative to the level of expression of a native yeast CD nucleic acid sequence. In a specific embodiment, the sequence of the present invention differs from the yeast CD encoding sequence at a total of 91 positions

(each of the 91 nucleotide changes being indicated by a box in Fig.

1 ).

It will be appreciated from a reading of this disclosure that not all 91 positions within the protein coding region, as noted in Fig. 1 , need be changed in order to obtain a CD encoding sequence in accordance with the invention that is sufficiently well expressed in tumor cells (eg human tumor cells) to be suitable for use in GDEPT or VDEPT. It will also be well understood by persons skilled in the art that minor modifications or alterations to the sequence shown in SEQ ID NO. 4 can be effected without substantially affecting CD activity or expression in mammalian cells. Such sequences which have substantial identity to SEQ ID NO. 4 and include up to about 5% of sequence mutations relative to SEQ ID NO. 4, are also considered to fall within the scope of the present invention.

A CD encoding sequence of the invention can be produced using, for example, the method of Sethna et al (J. Virol. 65:320 (1991 )). In accordance with this method, overlapping oligonucleotides approximately 75 bases long are chemically synthesized. Equimolar quantities of each oligonucleotide are then annealed, ligated and cloned into an appropriate vector. One chemically synthesized CD (CS-CD) of the invention is shown in Fig. 2. The GC content of this CD is about 54.8%. The CD sequences of the invention (or fragment thereof encoding an amino acid sequence that efficiently catalyzes 5-FC to 5-FU) can be incorporated into a recombinant molecule, including an expression cassette. Expression cassettes themselves are well known in the art of molecular biology. Such an expression cassette contains all essential DNA sequences required for expression of the heterologous enzyme in a mammalian cell. For example, an expression cassette will contain a molecular chimaera containing the coding sequence for CD of the invention, an appropriate polyadenylation signal for a mammalian gene (i.e., a polyadenylation signal that will function in a mammalian cell), and suitable 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 organization. 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 (UPEs). The UPEs are a principle target for the interaction with sequence- specific nuclear transcriptional factors. The activity of promoter sequences is 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 organization 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) enables 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. Examples include: i) the albumin (ALB) and alpha- fetoprotein (AFP) transcriptional regulatory sequence (for example, the promoter and enhancer) specific for normal hepatocytes and transformed hepatocytes, respectively, ii) the transcriptional regulatory sequence for carcinoembryonic antigen (CEA) for use in colorectal carcinoma, metastatic colorectal carcinoma, and hepatic colorectal metastases, transformed cells of the gastrointestinal tract, lung, breast and other tissues, iii) the transcriptional regulatory sequence for tyrosine hydroxylase, choline acetyl transferase, or neuron specific enolase for use in neuroblastomas, iv) the transcriptional regulatory sequence for glial fibro acidic protein for use in gliomas, and v) the transcriptional regulatory sequence for insulin for use in tumors of the pancreas. Further examples include the transcriptional regulatory sequence specific for gama-glutamyltranspeptidase for use in certain liver tumors and dopa decarboxylase for use in treating certain tumors of the lung.

In addition, the transcriptional regulatory sequences from certain oncogenes may be used as these are expressed predominantly in certain tumor types. Examples of these include the HER-2/neu oncogene regulatory sequence, which is expressed in breast tumors, and the regulatory sequence specific for the N- myc oncogene for neuroblastomas.

The ALB and AFP genes exhibit extensive homology with regard to nucleic acid sequence, gene structure, amino acid sequence, and protein secondary folding (for review see Ingram et al, Proc. Natl. Acad. Sci. USA 78: 4694 (1981 )). These genes are independently but reciprocally expressed in ontogeny. In normal development, ALB transcription is initiated shortly before birth and continues throughout adulthood. Transcriptional expression of ALB in the adult is confined to the liver. AFP is normally expressed in fetal liver, the visceral endoderm of the yolk sac, and the fetal gastrointestinal tract, but declines to undetectable levels shortly after birth and is not significantly expressed in nonpathogenic or nonregenerating adult liver or in other normal adult tissue. However, AFP transcription in adult liver often increases dramatically in HCC. In addition, AFP transcription may also be elevated in nonseminomatous and mixed carcinoma of the testis, in endodermal sinus tumors, in certain teratocarcinomas, and in certain gastrointestinal tumors. Liver-specific expression of AFP and ALB is the result of interactions of the regulatory sequences of their genes with trans-activating transcriptional factors found in nuclear extracts from liver.

Several mammalian ALB and AFP promoter and enhancer sequences have been identified (for review see Pinkert et al,

Genes Dev. 1 :268 (1987); Hammer et al, Science 235:53 (1987); Wantanabe et al, J. Biol. Chem. 262:4812 (1987)). These sequences enable the selective and specific expression of genes in liver hepatocytes (normal and transformed) and hepatomas, respectively. (See European Patent Publication No.729, 515, and references cited therein, for further details regarding ABL anf AFP promoter and enhancer sequences.)

Carcinoembryonic antigen (CEA) is a tumor-associated marker that is expressed in a large percentage of primary and metastatic CRC cells and is widely used as an important diagnostic tool for postoperative surveillance, chemotherapy efficacy determinations, immunolocalization and immunotherapy. By placing the expression of the CD encoding sequence of the invention under the transcriptional control of the CRC-associated marker gene, CEA, the nontoxic compound, 5-FC, can be metabolically activated to 5-FU selectively in CRC cells (for example, hepatic CRC cells). An advantage of this system is that the generated 5-FU can diffuse out of the cells in which it was generated and kill adjacent tumor cells which did not incorporate the CD gene of the invention. (See European Patent Publication

No. 729,515 for further details relating to CEA enhancer and promoter sequences.)

The promoter and enhancer sequences preferably are selected from the transcriptional regulatory sequence for one of albumin (ALB),alpha-fetoprotein (AFP), carcinoembryonic antigen

(CEA), cytomegalovirus(CMV), tryrosine hydroxylase, choline acetyl transferase, neuron-specific enolase, glial fibro acidic protein, insulin or gama-glutamyltranspeptidase, dopa- decarboxylase, HER-2/neu or N-myc oncogene or other suitable genes such as cytomegalovirus (CMV), SV40 or actin. Most preferably, the regulatory sequence for ALB or AFP is used to direct liver specific or hepatoma-specific expression, respectively, and the regulatory sequence for CEA is used to direct colorectal carcinoma, metastatic colorectal carcinoma (eg, hepatic colorectal carcinoma metastases) specific expression. The regulatory sequence for ALB or AFP can also be used to direct colorectal carcinoma or metastatic colorectal carcinoma (e.g., hepatic colorectal carcinoma metastases) specific expression. Furthermore, the regulatory sequence for CEA can also be used to direct liver-specific or hepatoma-specific expression.

The molecular chimera of the invention can be prepared utilizing standard recombinant techniques. These molecular chimaeras can be delivered to the target tissue or cells by a delivery system. For administration to a host (eg, a mammal, preferably a human), it is necessary to provide an efficient in vivo delivery system that stably incorporates the molecular chimaera into the cells. Known methods include techniques of calcium phosphate transfection, electroporation, microinjection, liposomal transfer, ballistic barrage, DNA viral infection or retroviral infection as well as other techniques which would be well known by persons skilled in the art. For a review of this subject, see Biotechniques 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 et al, Mol. Cell. Biol. 6:2895 (1986)). Essentially, retroviral shuttle vectors (retroviruses comprising molecular chimaeras 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 (e.g., gn, p_ol and env) removed from the vectors and the DNA sequence of interest (the CD encoding sequence of the invention) is inserted. The vectors also contain appropriate retroviral regulatory sequences for viral encapsulation, 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 can also be used as delivery systems.

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.

The advantages of a retroviral-mediated gene transfer system are the high efficiency of 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 little 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 which includes the CD encoding sequence of the invention and a 3' viral LTR sequence. In a preferred embodiment, and to help eliminate non- tissue-specific expression of the molecular chimaera, the molecular chimaera is placed in opposite transcriptional orientation to the 5' retroviral LTR. In addition, a dominant selectable marker gene may also be included that is transcriptionally driven from the 5' LTR sequence. Such a dominant selectable marker gene may be the bacterial neomycin-resistance gene NEO (aminoglycoside 3'phosphotransferase type II), which confers on eukaroytic cells resistance to the neomycin analogue Geneticin antibiotic G418 sulphate; registered trademark of GIBCO). The NEO gene aids in the selection of packaging cells that contain these sequences.

The retroviral vector is preferably based on the Moloney murine leukemia virus but it will be appreciated that other vectors may be used. Vectors containing a NEO gene as a selectable marker have been described, for example, the N2 vector (Eglitis et al, Science 230:1395 (1985)).

A theoretical problem associated with retroviral shuttle vectors is the potential of retroviral 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. SIN vectors are self-inactivating vectors that 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 et al, Biotechniques

4:504 (1986)).

Since the parental retroviral gag, pol, and env genes have been removed from these shuttle vectors, a helper virus system may be utilized to provide the gag, pol, and env retroviral gene products in trans to package or encapsulate the retroviral vector into an infective virion. This is accomplished by utilizing specialized "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 encapsulated into an infectious viral particle.

The present invention also relates to a method of producing infective virions 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. In addition to retroviral-mediated gene delivery of the chimeric, artificial, therapeutic gene, other gene delivery systems known to those skilled in the art can be used in accordance with the present invention. These other gene delivery systems include other viral gene delivery systems known in the art, such as the adenovirus delivery systems.

Non-viral delivery systems can be utilized in accordance with the present invention as well. For example, liposomal delivery systems can deliver the therapeutic gene to the tumor site via a liposome. Liposomes can be modified to evade metabolism and/or to have distinct targeting mechanisms associated with them. For example, liposomes which have antibodies incorporated into their structure, such as antibodies to CEA, can have targeting ability to CEA-positive cells. This will increase both the selectivity of the present invention as well as it's ability to treat disseminated disease (metastasis).

Another gene delivery system which can be utilized according to the present invention is receptor-mediated delivery, wherein the gene of choice is incorporated into a ligand which recognizes a specific cell receptor. This system can also deliver the gene to a specific cell type. Additional modifications can be made to this receptor-mediated delivery system, such as incorporation of adenovirus components to the gene so that the gene is not degraded by the cellular lysosomal compartment after internalization by the receptor.

The infective virion or the packaging cell line according to the invention may be formulated by techniques well known in the art and may be presented as a formulation (composition) with a pharmaceutically acceptable carrier therefor. Pharmaceutically acceptable carriers, in this instance physiologic aqueous solutions, may comprise 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 or packaging cell line may be a solution or suspension in such a vehicle. Stabilizers and antioxidants and/or other excipients may also be present in such pharmaceutical formulations (compositions), which may be administered to a mammal by any conventional method (e.g., oral or parenteral routes). In particular, the infective virion may be administered by intravenous or intra-arterial infusion. In the case of treating HCC or hepatic metastatic CRC, intra-hepatic arterial infusion may be advantageous. The packaging cell line can be administered directly to the tumor or near the tumor and thereby produce infective virions directly at or near the tumor site. Although any suitable compound that can be selectively converted to a cytotoxic or cytotostatic metabolite by the CD of the invention can be utilised, the preferred compound for use in accordance with the invention is 5-FC, in particular, in the treatment of cancers capable of expressing CD. In particular for use in treating hepatocellular carcinoma (HCC), colorectal carcinoma(CRC), metastatic colorectal carcinoma, or hepatic CRC metastases. Any agent that can potentiate the antitumor effects of 5-FU can also potentiate the antitumor effects of 5-FC) since, when used according to the present invention, 5-FC is selectively converted to 5-FU. According to another aspect of the present invention, agents such as leucovorin and levemisol, which can potentiate the antitumor effects of 5-FU, can also be used in combination with 5-FC when 5-FC is used according to the present invention. Other agents which can potentiate the antitumor effects of 5-FU are agents which block the metabolism of 5-FU. Examples of such agents are 5-substituted uracil derivatives, for example, 5- ethynyluracil and 5-bromvinyluracil (PCT/GB91/01650(WO 92/04901 ); Cancer Research 46:1094 (1986)). The present invention further includes the use of agents which are metabolised in vivo to the corresponding 5-substituted uracil derivatives described hereinbefore (see Biochem. Pharmacol. 38: 2885 (1989)) in combination with 5-FC when 5-FC is used according to the present invention. Two significant advantages of the enzyme/prod rug combination of CD/5-fluorocytosine and further aspects of the invention are the following:

1. The metabolic conversion of 5-FC by CD produces 5-FU, the drug of choice in the treatment of many different types of cancers, such as colorectal carcinoma.

2. The 5-FU that is selectively produced in one cancer cell can diffuse out of that cell and be taken up by both non-facilitated diffusion and facilitated diffusion into adjacent cells. This produces a neighboring cell killing effect. This neighbor cell killing effect alleviates the necessity for delivery of the therapeutic molecular chimera to every tumor cell. Rather, delivery of the molecular chimera to a certain percentage of tumor cells can produce the complete eradication of all tumor cells. The amounts and precise regimen in treating a mammal will 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. However, for HCC or hepatic metastatic CRC, an intrahepatic arterial infusion of the artificial infective virion at a titer of between 2 x 10⁵ and 2 x 10⁷ colony forming units per ml (CFU/ml) infective virions is suitable for a typical tumor. Total amount of virions infused will be dependent on tumor size and are preferably given in divided doses. Prodrug treatment - Subsequent to infection with the infective virion, certain cytosine compounds (prodrugs of 5-FU) are converted by CD to cytotoxic or cytostatic metabolites (eg 5-FC is converted to 5-FU) in the target cells. The abovementioned prodrug compounds are administered to the host (eg, a mammal, preferably, a human) between six hours and ten days, preferably between one and five days, after administration of the infective virion.

The dose of compound (eg, 5-FC) to be given will advantageously be in the range 10 to 500 mg per kg body weight of recipient per day, preferably 50 to 500 mg per kg bodyweight of recipient per day, more preferably 50 to 250 mg per kg bodyweight of recipient per day, and most preferably 50 to 150 mg per kg body weight of recipient per day. The modes of administration of 5-FC in humans are well known to those skilled in the art. Oral administration and/or constant intravenous infusion of 5-FC are anticipated by the instant invention to be preferable. The doses and mode of administration of leucovorin and levemisol to be used in accordance with the present invention are also well known or readily determined by clinicians skilled in the art of oncology. The dose and mode of administration of the 5-substituted uracil derivatives can be determined by the skilled oncologist. Preferably, these derivatives are given by intravenous injection or orally at a dose of between 0.01 to 50 mg per kilogram body weight of the recipient per day, particularly 0.01 to 10 mg per kilogram body weight per day, and more preferably 0.01 to 0.4 mg per kilogram bodyweight per day depending on the derivative used. An alternative preferred administration regime is 0.5 to 10 mg per kilogram body weight of recipient once per week.

The following non-limiting Example describes certain aspects of the invention in greater detail. (See also Kievit et al, Cancer Research 59:1417-1421 (1999)).

Example 1

Expression of CD Gene in Human Tumor Cell Lines

Expression of the E. coli and yeast CD genes and the CS- CD of Fig. 2 in human lung tumor cell lines A549 and H460 following lipofectamine-mediated gene transfer was demonstrated by evaluating cell lysates for functional CD, as manifest by the conversion of [³H]5FC to [³H]5FU in a time- and dose-dependent fashion. There was a similar dose-dependent increase in enzyme activity in H460 cells transfected with increasing amounts of the

CS-CD and the E. coli and yeast CD genes (Fig. 3). The levels of CD in H460 cells transfected with CS-CD gene were on the average 330 ± 20 (n = 4)- and 540 ± 120 (n = 4)-times higher than in the cells transfected with equal amounts of E. coli and yeast CD DNA (Fig. 3). A similar increase in the specific activity of the enzyme in A549 cells transfected with CS-CD DNA compared to yeast and E. coli DNA was also observed (Fig. 4). Thus, the specific activity of CD was consistently several hundred-fold higher in the cells transfected with the CD DNA of the invention than in the human tumor cells transfected with either E. coli or yeast CD.

The increased expression of the CS-CD compared to the yeast CD in the lung tumor cell lines was further confirmed by measuring the steady state levels of protein by Western blot analysis. The yeast CD protein from cells transfected with the CD DNA of the invention or the yeast CD DNA was resolved by polyacrylamide gel electrophoresis and transferred to PVDF membranes. The protein was detected by an antibody specific to the yeast CD. Western blots or the membranes were developed using the ECL western blotting system (Amersham Pharmacia Biotech) according to the manufacture's specifications. The amount of protein was quantitated by Storm (Molecular Dynamics, Calif.) a Phosphor-lmager using ImageQuant software. The steady state levels of yeast CD were several hundred-fold higher in cells transfected with the CS-CD than the yeast CD.

The expression of the CD DNA of the invention was also substantially higher in tumor types other than the lung tumor cell lines. The expression of the CD DNA of the invention (CS-CD) was compared with the CD gene from E. coli in a panel of tumor cell lines as shown in Table I. The specific activity for the CS-CD of the invention was 20-800 times higher than that of the bacterial gene in the different tumor cell lines tested.

Table I Expression of CS-CD and E. Coli CD in different human cell-lines

Cells were transfected with DNA and extracts were prepared as described in description of Figure 3.

Replicates of four to eight assays were used to determine SE.

*Enzymes activity is expressed as μmol product formed at 37°C/min/mg protein. Example 2

Chimeras of Yeast CD and CS-CD

A total of 91 changes were made in the yeast cytosine deaminase gene to obtain the CS-CD shown in SEQ ID NO:4. Preliminary experiments have been carried out to demonstrate which of these mutations are needed to attain the desired effect (see Figs. 5A-5D). Four DNA constructs have been prepared

(YCDM #A (SEQ ID NO:5), YCDM #B (SEQ ID NO:6), YCDM #C (SEQ ID NO:7), YCDM #D (SEQ ID NO:8)). Each is a chimera of the yeast CD of SEQ ID NO:1 and the cytosine deaminase of the invention (CS-CD) (SEQ ID NO:4). The four mutants encompass the whole gene. In the cloning vectors used in the preliminary studies, mutants YCDM #B and YCDM #C were unstable and thus it was not possible to characterize them further. In addition, the subcloning of YCDM #D into a vector in the preliminary studies resulted in a change in reading frame and a premature stop that shortened the protein by six amino acids and altered the last three amino acids of the protein. When expressed in a human lung tumor cell line, no detectable enzyme activity was observed (Table II).

In mutant YCDM #A, 18 changes at 5' end of the gene were replaced with the parent yeast base pairs and 73 changes at the 3' end were retained. YCDM #A was expressed in a human lung tumor cell line and the results are shown in Table II. The specific activity of the enzyme expressed in cells transfected with YCDM #A was very similar to that observed in the cells transfected with the parent yeast CD. The cells transfected with CD gene of the invention (CS-CD) had about 100 times more enzyme activity than the cells transfected with the YCDM #A and the parent yeast CD. While these preliminary data suggest that the 18 changes at the 5' end of the gene are essential for the improved activity of the CS-CD gene, they do not indicate that the remaining 73 changes are not important for the improved activity.

Table II Expression of CS-CD, yeast CD, E. coli CD, YCDM #A and YCDM #D in human lung large cell carcinoma (H460) cells.

Ceil Line CD

Enzyme Activity + SE*

Yeast CD 0.25 + 0.003

CS-CD 27.33 + 5.29

E. coli CD 0.018 + 0.002

YCDM #A 0.247 + 0.035

YCDM #D ND

Cells were transfected with DNA and extracts were prepared as described in description of Figure 3.

Replicates of six to nine assays were used to determine SE.

^*Enzyme activity is expressed as μmol product formed at 37°C/min/mg protein.

ND, not detectable.

Example 3

Mutants of Protein Encoded by CS-CD

Four mutants YCDM #1 (SEQ ID NO:9), YCDM #2 (SEQ ID NO:11 ), YCDM #3 (SEQ ID NO:13), and YCDM #4 (SEQ ID

NO: 15), have been generated that encode proteins with (SEQ ID NOs: 10, 12, 14 and 16, respectively) changes in specific amino acids in the CS-CD protein sequence. (See also Figs. 6A-6D). The mutants were generated by a method that was used to produce the CD DNA of the invention (Sethna et al, J. Virol. 65:320

(1991 )).

The expression of the mutants was compared with the CD DNA of the invention in the lung tumor cell line (H460). The specific activity of CD in H460 cells transfected with the four mutants were orders of magnitude lower than the cells transfected with CD gene of the invention (CS-CD). In preliminary studies, the CD specific activity from YCDM #1 , 2 and 4 was 800-fold less than CS-CD. However, CD specific activity expressed by YCDM #3 was only 100 fold less than the CS-CD.

The reduced activity of the mutant genes could result from either a reduction in the total amount of the protein synthesized or from a reduced catalytic activity of the protein. It appears that the reduced activity of the mutants is due to the reduced expression of the mutant genes. The steady state levels of the yeast CD protein in the cells transfected with the mutant genes were measured by Western blot analysis as described previously. The steady state levels of the protein made in the cells transfected with the mutant genes were significantly less than the protein levels in the cells transfected with CS-CD. A reasonable correlation between the enzyme specific activity and the steady state levels of the protein was observed.

* * * The entire contents of all documents cited above are incorporated in their entirety by reference. While the invention has been described in connection with what is presently considered to be the most practical and preferred embodiment, it is to be understood that the invention is not to be limited to the disclosed embodiment, but on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A nucleic acid sequence encoding cytosine deaminase (CD), wherein said nucleic acid sequence has a guanine:cytosine (G:C) content higher than that of a naturally occurring CD encoding nucleic acid sequence.

2. The nucleic acid sequence according to claim 1 wherein said CD is yeast CD.

3. The nucleic acid sequence according to claim 1 wherein said nucleic acid sequence comprises codons optimal for expression in a mammalian host cell.

4. The nucleic acid sequence according to claim 1 , which is between about 300 and about 500 times as efficient as an E. coli CD gene in converting 5-FC to 5-FU, in mammalian cells.

5. The nucleic acid sequence according to claim 1 wherein said nucleic acid is the same as or has substantial identity to the sequence of SEQ ID NO:4.

6. A molecular chimera comprising a transcriptional regulatory sequence (TRS) and the nucleic acid sequence of claim 1 operably linked thereto.

7. The chimera according to claim 6 wherein the TRS is selectively activated in a target mammalian cell.

8. The chimera according to claim 7 wherein the mammalian cell is a tumor cell.

9. The chimera according to claim 6 wherein the TRS comprises a promoter.

10. The chimera according to claim 9 wherein the promoter is selected from the group consisting of the promoters of albumin, alpha-fetoprotein, carcinoembyonic antigen, tyrosine hydroxylase, choline acetyl transferase, neuron-specific enolase, glial fibro acidic protein, insulin, gamma- glutamyltranspeptidase, dopa- decarboxylase, HER-2/neu and N-myc oncogenes.

11. A recombinant molecule comprising a vector and the chimera according to claim 6.

12. The molecule according to claim 11 wherein the vector is a viral vector.

13. The molecule according to claim 12 wherein said viral vector is a retroviral vector or an adenoviral vector.

14. The molecule according to claim 11 wherein said vector is a retroviral shuttle vector.

15. A packaging cell comprising the vector according to claim 14.

16. An infective virion comprising the chimera according to claim 6.

17. A pharmaceutical composition comprising the nucleic acid according to claim 1 and a pharmaceutically acceptable carrier.

18. A host cell comprising the nucleic acid according to claim 1.

19. The host cell according to claim 18 wherein the host cell is a mammalian cancer cell.

20. A method of treating a patient bearing a tumor comprising introducing into cells of said tumor the nucleic acid according to claim 1 under conditions such that said nucleic acid is expressed and said CD is thereby produced, and administering to said patient a nontoxic compound that is converted to a toxic compound by CD, wherein said nontoxic compound is administered in an amount such that sufficient toxic compound is produced by said CD to kill, or inhibit growth of, said cells.

21. A liposome comprising the nucleic acid of claim 1.

22. Use of the nucleic acid sequence as claimed in claim 1 in a method of treatment of cancer in a patient.

23. A nucleic acid sequence as claimed in any one of claims 1 to 5, a chimera as claimed in any one of claims 6 to 19, a molecule according to any one of claims 11 to 14, a virion according to claim 16 or a composition according to claim 17 for use in therapy.

24. Use of a nucleic acid sequence as claimed in any one of claims 1 to 5, a chimera as claimed in any one of claims 6 to 19, a molecule according to any one of claims 11 to 14, a virion according to claim 16 or a composition according to claim 17 in the manufacture of a medicament for the treatment of cancer.