WO1996036365A1 - Gene therapy of hepatocellular carcinoma through cancer-specific gene expression - Google Patents

Abstract

A DNA virus vector, such as, for example, an adenoviral vector which includes at least one DNA sequence encoding at least one agent capable of providing for the inhibition, prevention, or destruction of the growth of tumor cells, such as a negative selective marker (e.g., Herpes Simplex Virus thymidine kinase), and a liver tumor cell specific promoter (e.g., the alphafetoprotein promoter) controlling the at least one DNA sequence encoding the at least one agent. Such a vector may transduce hepatocellular carcinoma cells in a host, whereby such cells are killed upon the administration of an interaction agent or prodrug (e.g., gancyclovir) to a host.

Description

GENE THERAPY OF HEPATOCELLULAR CARCINOMA THROUGH CANCER-SPECIFIC GENE EXPRESSION

This invention relates to the treatment of hepatocellular carcinoma. More particularly, this invention relates to the treatment of hepatocellular carcinoma with DNA virus vectors, such as, for example, adenoviral vectors including a DNA sequence encoding an agent capable of providing for the inhibition, prevention, or destruction of the growth of hepatocellular carcinoma cells and a promoter controlling the DNA sequence encoding said agent, said promoter being a liver tumor cell specific promoter. BACKGROUND OF THE INVENTION

Hepatocellular carcinoma is one of the most common cancers in the world. Only a minority of patients are curable by removal of the tumor either by resection or transplantation. (Tang, et al . , Cancer, Vol. 64, pgs . 536- 541 (1989) ; Yamana a, et al . , Cancer, Vol. 65, pgs. 1104-1110 (1990) ; Iwatsuki, Ann. Surσ.. Vol. 214, pgs. 221-229 (1991)) . For the majority of patients, the current treatments remain unsatisfactory, and the prognosis is poor. One of the characteristics of hepatocellular carcinoma in that most patients have an elevated serum level of alphafetoprotein (or AFP) . (Alpert, et al . , Hepatocellular Carcinoma, pgs. 353- 367, Okuda, et al . , eds . , John Wiley & Sons, Inc., New York (1976) ) . The serum concentration generally tends to stabilize or increase gradually with progression of disease, and high levels of alphafetoprotein are found frequently in patients with advanced hepatocellular carcinoma. The serum AFP levels in the patients appear to be regulated by AFP expression in hepatocellular carcinoma but not in surrounding normal liver. (Engelhardt, et al . , Int. J. Cancer, Vol. 7, pgs. 198-206 (1971) ; Peng, et al . , Hepatolocrv, Vol. 17, pgs. 35-41 (1993) ) .

Retrovirus-mediated transfer of the Herpes Simplex Virus thymidine kinase (HSV-TK) gene has been used to confer cytotoxic sensitivity to the nucleoside analogue gancyclovir (GCV) , in a variety of tumor cells in vi tro and in vivo . (Moolten, et al . , Cancer Research, Vol. 46, pgs. 5276-5281 (1986) ; Borrelli, et al . , Proc. Nat. Acad. Sci., Vol. 85, pgs. 7572-7576 (1988) ; Moolten, et al . , J. Nat. Cancer Inst . , Vol. 82, pgs. 297-300 (1990); Ezzedine, et al . , New Biologist, Vol. 3, pgs. 608-614 (1991); Culver, et al . , Science. Vol. 256, pgs. 1550-1552 (1992)) . HSV-TK converts gancyclovir into a phosphorylated compound that acts as a chain terminator in DNA synthesis, killing HSV-TK-containing cells. (St. Clair, et al . , Antimicrob. Agents Chemother. , Vol. 31, pgs. 844-849 (1987)) . This vector system, however, is limited by relatively low viral titer -and low target cell transduction frequency.

Recombinant adenoviruses can be produced in high titers and transduce efficiently a variety of cells, and allowing a high transduction frequency in a solid tumor. (Brody, et al . , Human Gene Therapy. Vol. 5, pgs. 437-447 (1994); Chen, et al . , Proc. Nat. Acad. Sci., Vol. 91, pgs. 3054-3057 (1994) ; Fujiwara, et al . , Cancer Research, Vol. 54, pgs. 2287-2291 (1994); Wills, et al . , Human Gene Therapy. Vol. 5, pgs. 1079-1088 (1994) ; Liu, et al . , Cancer Research, Vol. 54, pgs. 3662-3667 (1994)) . Thus, adenoviral vectors have been used in gene therapy for the treatment of solid tumors . Adenoviral vectors, however, can infect normal cells as well as tumor cells, which may cause toxicity in normal cells or tissues. If HSV-TK expression can be limited to tumor cells, however, by employing a tumor specific promoter, this problem may be overcome. SUMMARY OF THE INVENTION

The present invention is directed to the treatment of hepatocellular carcinoma by administering to a host suffering from hepatocellular carcinoma a DNA virus vector which includes a DNA sequence encoding an agent capable of providing for the inhibition, prevention, or destruction of the growth of hepatocellular carcinoma cells and a liver tumor cell specific promoter controlling the DNA sequence encoding the agent. In one embodiment, the DNA virus vector is an adenoviral vector. BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described with respect to the drawings, wherein:

Figure 1 is a schematic of the construction of plasmid pHR;

Figure 2 is a schematic of the construction of an adenoviral vector including an ITR, an encapsidation signal, a Rous Sarcoma Virus promoter, and an adenoviral tripartite leader (TPL) sequence;

Figure 3 is a schematic of the construction of pAvS6;

Figure 4 is a map of plasmid pAvS6;

Figure 5 is a map of plasmid pAvS6-nLacZ;

Figure 6 is a schematic of the construction of

AvlLacZ4

Figure 7 is a schematic of the construction of plasmid pAvSδ.TKl;

Figure 8 is a map of plasmid pAvS12;

Figure 9 is a map of pAvSCMV;

Figure 10 is a schematic of the construction of pAvSlO.TKl; Figure 11 is a schematic of the construction of plasmid pAvS20.TKl;

Figure 12 is a schematic of the construction of plasmid pAvS21.TKl;

Figure 13 is a graph of the survival of HuH7 cells infected with AvlAFPTKl in vi tro, followed by gancyclovir treatment;

Figure 14 is a graph of the survival of SK-Hep-1 cells infected with AvlAFPTKl in vi tro, followed by gancyclovir treatment;

Figure 15 is a graph of the survival of HuH7 cells infected with AvlTKl in vi tro, followed by gancyclovir treatment;

Figure 16 is a graph of the survival of SK-Hep-1 cells infected with AvlTKl in vi tro, followed by gancyclovir treatment;

Figure 17 is a graph of the survival of HuH7 cells infected with AvlLacZ4 in vi tro, followed by gancyclovir treatment;

Figure 18 is a graph of the survival of SK-Hep-1 cells infected with AvlLacZ4 in vi tro, followed by gancyclovir treatment;

Figure 19 is a graph of tumor size in mice having tumors formed of HuH7 cells, following administration to the mice of AvlAFPTKl, AvlTKl, or AvlLacZ4, or buffer, with or . without gancyclovir treatment; and

Figure 20 is a graph of tumor size in mice having tumors formed of SK-Hep-1 cells, following administration to the mice of AvlAFPTKl, AvlTKl, or AvlLacZ4, or buffer, with or without gancyclovir treatment.

In accordance with an aspect of the present invention, there is provided a DNA virus vector which includes (i) at least one DNA sequence encoding an agent capable of providing for the inhibition, prevention, or destruction of the growth of tumor cells; and (ii) a promoter specific for liver tumor cells controlling the DNA sequence encoding the agent.

The term "promoter specific for liver tumor cells" as used herein, means that the promoter is operative in most liver tumor cells and is not operative in non-fetal normal liver cells. Genes under control of such promoter will be expressed in liver tumor cells.

DNA virus vectors which may be employed include, but are not limited to, adenoviral vectors; members of the parvorius family of vectors (including adeno-associated virus (AAV) vectors) ; and DNA tumor virus vectors, such as, for example, Herpes Virus vectors and vectors dervied from Epstein-Barr Virus (EBV) which have been engineered to render such viruses replication-defective.

In one embodiment, the DNA virus vector is an adenoviral vector.

The adenoviral vector which is employed may, in one embodiment, be an adenoviral vector which includes essentially the complete adenoviral genome (Shenk, et al . , Curr. Top. Microbiol. Immunol.. 111(3) : 1-39 (1984)) . Alternatively, the adenoviral vector may be a modified adenoviral vector in which at least a portion of the adenoviral genome has been deleted.

In one embodiment, the adenoviral vector comprises an adenoviral 5' ITR; an adenoviral 3' ITR; an adenoviral - encapsidation signal; and at least one DNA sequence encoding the at least one agent . The vector is free of at least the majority of adenoviral El and E3 DNA sequences, but is not free of all of the E2 and E4 DNA sequences, and DNA sequences encoding adenoviral proteins promoted by the adenoviral major late promoter.

In still another embodiment, the gene in the E2a region that encodes the 72 kilodalton binding protein is mutated to produce a temperature sensitive protein that is active at 32°C, the temperature at which the viral particles are produced. This temperature sensitive mutant is described in Ensinger, et al . , J. Virology. 10:328-339 (1972), Van der Vliet, et al., J. Virology. 15:348-354 (1975) , and Friefeld, et al . , Virolog^y. 124:380-389 (1983) .

Such a vector, in a preferred embodiment, is constructed first by constructing, according to standard techniques, a shuttle plasmid which contains, beginning at the 5' end, the "critical left end elements," which include an adenoviral 5' ITR, an adenoviral encapsidation signal, and an Ela enhancer sequence; a promoter; a multiple cloning site; a poly A signal; and a DNA segment which corresponds to a segment of the adenoviral genome. The vector also may contain a tripartite leader sequence. The DNA segment corresponding to the adenoviral genome serves as a substrate for homologous recombination with a modified or mutated adenovirus, and such sequence may encompass, for example, a segment of the adenovirus 5 genome no longer than from base 3329 to base 6246 of the genome. The plasmid may also include a selectable marker and an origin of replication. The origin of replication may be a bacterial origin of replication. Representative examples of such shuttle plasmids include pAvS6, shown in Figure 4. The at least one DNA sequence encoding the at least one agent then may be inserted into the multiple cloning site. The liver tumor cell specific promoter controlling the at least one DNA sequence encoding • the at least one agent may be inserted into the multiple cloning site, or, alternatively, the promoter may be inserted into another portion of the plasmid. One may amplify the expression of the DNA encoding the agent by adding to the plasmid increased copies of the DNA encoding the agent .

This construct is then used to produce an adenoviral vector. Homologous recombination is effected with a modified or mutated adenovirus in which at least the majority of the El and E3 adenoviral DNA sequences have been deleted. Such homologous recombination may be effected through co- transfection of the plasmid vector and the modified adenovirus into a helper cell line, such as 293 cells (ATCC No. CRL 1573⁾ , by CaP0₄ precipitation. Upon such homologous recombination, a recombinant adenoviral vector is formed that includes DNA sequences derived from the shuttle plasmid between the NotI site and the homologous recombination fragment, and DNA derived from the El and E3 deleted adenovirus between the homologous recombination fragment and the 3' ITR.

In one embodiment, the homologous recombination fragment overlaps with nucleotides 3329 to 6246 of the adenovirus 5 (ATCC VR-5) genome.

Through such homologous recombination, a vector is formed which includes an adenoviral 5' ITR, an adenoviral encapsidation signal; an Ela enhancer sequence; a liver tumor cell specific or hepatocellular carcinoma specific promoter; the at least one DNA sequence which encodes the at least one agent; a poly A signal; adenoviral DNA free of at least the majority of the El and E3 adenoviral DNA sequences; and an adenoviral 3' ITR. The vector also may include a tripartite leader sequence. This vector may then be transfected into a helper cell line, such as the 293 helper cell line, which will include the Ela and Elb DNA sequences, which are necessary for viral replication, and to generate infectious adenoviral particles.

The adenoviral vector particles are administered to a host such that the adenoviral vector particles transduce hepatocellular carcinoma cells . The adenoviral vector particles may be administered systemically, such as by intraperitoneal administration, intravascular administration, including intravenous (e.g., by administration into the hepatic portal vein) , or intraarterial administration (i.e., administration into the hepatic artery) . Alternatively, the adenoviral vector particles may be administered directly to a tumor formed of hepatocellular carcinoma cells.

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3UBSTITUTE SHEET (RULE 26) The adenoviral particles are administered in an amount effective to produce a therapeutic effect in a host. The host may be a mammalian host, including human and non-human primate hosts. In one embodiment, the adenoviral particles are administered in an amount of at least 10⁷ plaque forming units (pfu) , and in general such amount does not exceed about 10¹³ pfu, and preferably is from about 10⁸ pfu to about 10" pfu. The exact dosage of adenoviral vector particles to be administered is dependent on a variety of factors, including the age, weight, and sex of the patient to be treated, and the nature and extent of the hepatocellular carcinoma to be treated. The adenoviral particles may be administered as part of a preparation having a titer of adenoviral partic^s of at least 10¹⁰ pfu/ml, and in general not exceeding 10¹² pfu/ml. The adenoviral particles may be administered in combination with a pharmaceutically acceptable carrier in a volume up to 5 ml.

The adenoviral vector particles may be administered in combination with a pharmaceutically acceptable carrier suitable for administration to a patient, such as, for example, a liquid carrier such as a saline solution, protamine sulfate (Elkins-Sinn, Inc., Cherry Hill, N.J.), water, aqueous buffers such as phosphate buffers and Tris buffers, or Polybrene (Sigma Chemical, St. Louis, MO) . The selection of a suitable pharmaceutical carrier is deemed to be apparent to those skilled in the art from the teachings contained herein.

Liver tumor cell specific promoters which may be employed include, but are not limited to, the alphafetoprotein promoter, the RGTP promoter, the IGF-II promoter (van Dijk, Mol . Cell . Endocrinol . , Vol. 88, pgs. 175-185 (1992)) ; the bFGF promoter (Veba, et al. , Proc. Nat. Acad. Sci. , Vol. 91, pg. 9009 (1994)); and the hepatitis x dependent specific promotors (Schluter, Oncogene, Vol. 9, pgs. 3335-3344 (1994)) . In one embodiment, the promoter is the alphafetoprotein promoter.

Agents which are capable of providing for the inhibition, prevention, or destruction of the growth of the hepatocellular carcinoma cells include, but are not limited to, negative selective markers; toxins; antisense RNAs; ribozymes, intracellular antibodies; tumor suppressor proteins; and cytokines .

In one embodiment, the agent is a negative selective marker.

Negative selective markers which may be employed include, but are not limited to, thymidine kinase, such as Herpes Simplex Virus thymidine kinase, cytomegalovirus thymidine kinase, and varicella-zoster virus thymidine .nase; and cytosine deaminase.

Thus, upon transduction of the hepatocellular carcinoma cells with the adenoviral vector particles including a DNA sequence encoding a negative selective marker, and a liver tumor cell specific or hepatocellular carcinoma specific promoter controlling said DNA sequence encoding a negative selective marker, an interaction agent or prodrug is administered to the host. The interaction agent or prodrug interacts with the negative selective marker in order to prevent, inhibit, or destroy the growth of the hepatocellular carcinoma.

In one embodiment, the negative selective marker is a viral thymidine kinase selected from the group consisting of Herpes Simplex Virus thymidine kinase, cytomegalovirus thymidine kinase, and varicella-zoster virus thymidine kinase. When such viral thymidine kinases are employed, the interaction or chemotherapeutic agent or prodrug preferably is a nucleoside analogue, for example, one selected from the group consisting of gancyclovir, acyclovir, and l-2-deoxy-2- fluoro-3-D-arabinofuranosil-5-iodouracil (FIAU) . Such interaction agents or prodrugs are utilized efficiently by

^■ 9-

UBSTITUTE SHEET (RULE 26) the viral thymidine kinases as substrates, to produce a substance which is lethal to the hepatocellular carcinoma cells expressing the viral thymidine kinases, thereby resulting in inhibition of the growth of or the destruction of the tumor formed of hepatocellular carcinoma cells.

In another embodiment, the negative selective marker is cytosine deaminase. When cytosine deaminase is the negative selective marker, a preferred interaction agent or prodrug is 5-fluorocytosine. Cytosine deaminase converts 5- fluorocytosine to 5-fluorouracil, which is highly cytotoxic. Thus, the hepatocellular carcinoma cells which express the cytosine deaminase gene convert the 5-fluorocytosine to 5- fluorouracil to inhibit the growth of and/or destroy the tumor formed of hepatocellular carcinoma cells .

The interaction agent or prodrug is administered in an amount effective to inhibit, prevent, or destroy the growth of the tumor cells. For example, the interaction agent or prodrug may be administered in an amount from about 5 mg to about 15 mg/kg of body weight, preferably about 10 mg/kg, depending on overall toxicity to a patient. The interaction agent or prodrug may be administered in a single dose or in multiple doses. In one embodiment, the interaction agent or prodrug is administered twice per day in an amount of 5 mg/kg per dose.

When the adenoviral vectors including a polynucleotide . encoding a negative selective marker are administered as hereinabove described to a host, or administered directly to the tumor, a "bystander effect" may result, i.e., hepatocellular carcinoma cells of the tumor which were not originally transduced with the negative selective marker may be killed upon transfer of phosphorylated prodrug (e.g., gancyclovir) to neighboring cells. Although the scope of the present invention is not intended to be limited by any theoretical reasoning, the transformed tumor cells may be producing a diffusible form of the negative selective marker that either acts extracellularly upon the interaction agent or prodrug, or is taken up by adjacent, non-transformed tumor cells, which then become susceptible to the action of the interaction agent or prodrug. It also is possible that one or both of the negative selective marker and the interaction agent or prodrug are communicated between tumor cells .

In another embodiment, the agent is a toxin. Examples of toxins which may be employed include, but are not limited to, diphtheria toxin (Brietman, et al . , Mol . Cell . Biol . , Vol. 10, pgs. 474-479 (1990)) ; and Pseudomonas toxins.

In another embodiment, the agent is an antisense RNA. Antisense RNA sequences which may be encoded include, but are not limited to, antisense RNA sequences which bind to oncogenes . Such sequences include, but are not limited to, antisense RNA sequences which bind to the ras, bc!2, raf, or Bcr-Abl oncogenes .

In yet another embodiment, the agent is a ribozyme. Ribozymes which may be encoded include, but are not limited to, those which cleave mRNA of the oncogenes hereinabove described.

In another embodiment, the agent is an intracellular antibody. Such antibodies include, but are not limited to, antibodies which recognize liver tumor cell surface specific antigens.

In another embodiment, the agent is a tumor suppressor . protein. Examples of tumor suppressor proteins which may be encoded by the at least one DNA sequence include, but are not limited to, p53 protein; Rb protein; taxol; and vinblastine, as well as those described in Hartwell, et al. , Science, Vol. 266, pgs. 1821-1828 (1994) and in Levine, Am. Rev. Biochem. , Vol. 62, pgs. 623-651 (1993) .

In yet another embodiment, the agent is a cytokine. Examples of cytokines which may be encoded by the at least one DNA sequence include, but are not limited to, interleukins (including Interleukin-1, Interleukin-2, Interleukin-3 , Interleukin-4, Interleukin-6, Interleukin-7, Interleukin-10, and Interleukin-12) ; interferons (including Interferon-α, Interferon-3, and Interferon-γ) ; tumor necrosis factors, such as TNF-α; and colony stimulating factors such as GM-CSF, M-CSF, and G-CSF. Such cytokines may elicit an immune response against the hepatocellular carcinoma, thereby providing long-term immunity against hepatocellular carcinoma, and/or may provide a non-immune mediated anti- tumor effect as well. Such cytokines also are reviewed in Tepper, et al . , Human Gene Therapy, Vol. 5, pgs. 153-164 (1994); Columbo, et al . , Immunology Today, Vol. 15, pgs. 48- 51 (1994) ; Forni, et al. , Journal of Immunotherapy. Vol. 14, pgs. 310-313 (1993); and Pardol, Immunology Today, Vol. 14, pgs. 310-316 (1993) .

In one embodiment, the adenoviral vector may include more than one DNA sequence encoding more than one agent selected from those hereinabove described. In such an embodiment, each of the DNA sequences is under the control of the promoter specific for liver tumor cells. * In another embodiment, the DNA virus vector is a vector of the parvovirus family of vectors. Such vectors include, but are not limited to, adeno-associated virus vectors.

In yet another embodiment, the DNA virus vector is a DNA tumor virus vector which has been engineered to render the virus replication-defective. Such vectors include, but are not limited to, Herpes Virus vectors (including Herpes Virus 7, Herpes Virus 16, and Herpes Virus 18) , and vectors derived from Epstein-Barr Virus.

The above DNA viral vectors may be engineered according to techniques known to those skilled in the art to include the at least one DNA sequence encoding the at least one agent and the promoter specific for liver tumor cells controlling the at least one DNA sequence encoding the at least one agent . The DNA virus vectors (such as, for example, the adenovirus vectors) including the at least one DNA sequence encoding the at least one agent and the promoter controlling the at least one DNA sequence encoding the at least one agent may be employed in an animal model, wherein the DNA virus vector is administered to an animal in vivo. The animal then is evaluated for expression of the at least one agent in vivo in order to determine the effectiveness of the treatment in a human patient.

Alternatively, a genetic construct including the at least one DNA sequence encoding the at least one agent and the promoter specific for liver tumor cells controlling the at least one DNA sequence encoding the at least one agent may be encapsulated in a lipid vesicle, such as a liposome which is administered to a host in an amount effective to inhibit, prevent, or destroy the growth of hepatocellular carcinoma cells. The promoter and the at least one DNA sequence encoding the at least one agent may be in the form of linear DNA, or may be contained in an appropriate plasmid vector. Plasmid vectors which may be employed include, but are not limited to, plasmid vectors derived from adenoviruses such as those herein described; or plasmid vectors derived from other DNA viruses such as, for example, adeno-associated virus, Herpes Virus, or Epstein-Barr Virus.

The genetic construct including the at least one DNA sequence encoding the agent and the promoter specific for liver tumor cells controlling the at least one agent may be encapsulated within the lipid vesicle by means known to those skilled in the art. Such lipid vesicles then may be administered to a host in combination with an acceptable pharmaceutical carrier in an amount effective to inhibit, prevent, or destroy the growth of the cells of the hepatocellular carcinoma. EXAMPLES

The invention now will be described with respect to the following examples; however, the scope of the present invention is not intended to be limited thereby.

Example 1 The adenoviral vectors used in these examples (AvlLacZ4, AvlTKl, and AvlAFPTKl) are replication deficient Ela/Elb, E3 deletion mutants. These vectors were produced by recombination of the expression cassette with a cotransfected adenoviral genome in 293 cells for complementation of the El defect, thereby allowing virion production. Viral supernatants were harvested by 3 freeze-thaw cycles, followed by purification by ultracentrifugation through two cesium chloride gradients .

AvlLacZ4 is constructed from the adenoviral shuttle vector pAvS6. The construction of AvlLacZ4 is described in detail as follows: (i) Construction of pAvS6

The adenoviral construction shuttle plasmid pAvS6 was constructed in several steps using standard cloning techniques including polymerase chain reaction based cloning techniques. First, the 2913 bp Bglll, Hindlll fragment was removed from Ad-dl327 and inserted as a blunt fragment into the Xhol site of pBluescript II KS- (Stratagene, La Jolla, CA) (Figure 1) . Ad-dl327 (Thimmappaya, et al . , Cell, Vol. 31, pg. 543 (1983)) is identical to adenovirus 5 except that an Xbal fragment including bases 28591 to 30474 (or map units 78.5 to 84.7) of the adenovirus 5 genome, and which is located in the E3 region, has been deleted. The orientation of this fragment was such that the Bglll site was nearest the T7 RNA polymerase site of pBluescript II KS^" and the Hindlll site was nearest the -T3 RNA polymerase site of pBluescript II KS^". This plasmid was designated pHR. (Figure 1) . Second, the ITR, encapsidation signal, Rous Sarcoma Virus promoter, the adenoviral tripartite leader (TPL) sequence and linking sequences were assembled as a block using PCR amplification (Figure 2) . The ITR and encapsidation signal (sequences 1-392 of Ad-dl327 [identical to sequences from Ad5, Genbank accession #M73260] ) were amplified (amplification 1) together from Ad-dl327 using primers containing NotI or AscI restriction sites. The Rous Sarcoma Virus LTR promoter was amplified (amplification 2) ^•from the plasmid pRC/RSV (sequences 209 to 605; Invitrogen, San Diego, CA) using primers containing an AscI site and an Sfil site. DNA products from amplifications 1 and 2 were joined using the "overlap" PCR method (amplification 3) with only the NotI primer and the Sfil primer. Complementarity between the AscI containing end of each initial DNA amplification product from reactions 1 and 2 allowed joining of these two pieces during amplification. Next the TPL was amplified (amplification 4) (sequences 6049 to 9730 of Ad- dl327 [identical to similar sequences from Ad5, Genbank accession #M73260] ) from cDNA made from mRNA isolated from 293 cells infected for 16 hrs . with Ad-dl327 using primers containing Sfil and Xbal sites respectively. DNA fragments from amplification reactions 3 and 4 were then joined using PCR (amplification 5) with the NotI and Xbal primers, thus creating the complete gene block.

Third, the ITR-encapsidation signal-TPL fragment was then purified, cleaved with NotI and Xbal and inserted into the NotI, Xbal cleaved pHR plasmid. This plasmid was designated pAvS6A and the orientation was such that the NotI site of the fragment was next to the T7 RNA polymerase site (Figure 3) .

Fourth, the SV40 early polyA signal was removed from SV40 DNA as an Hpal-BamHI fragment, treated with T4 DNA polymerase and inserted into the Sail site of the plasmid pAvS6A- (Figure 3) to create pAvS6 (Figures 3 and 4.) (ii) Construction of AylLacZ .

The recombinant, replication-deficient adenoviral vector AvlLacZ4, which expresses a nuclear-targetable B- galactosidase enzyme, was constructed in two steps. First, a transcriptional unit consisting of DNA encoding amino acids 1 through 4 of the SV40 T-antigen followed by DNA encoding amino acids 127 through 147 of the SV40 T-antigen (containing the nuclear targeting peptide Pro-Lys-Lys-Lys-Arg-Lys-Val) , followed by DNA encoding amino acids 6 through 1021 of E. coli B-galactosidase, was constructed using routine cloning and PCR techniques and placed into the EcoRV site of pAvS6 to yield pAvS6-nlacZ (Figure 5) .

The infectious, replication-deficient, AvlLacZ4 was assembled in 293 cells by homologous recombination. To accomplish this, plasmid pAvS6-nLacZ was linearized by cleavage with Kpnl . Genomic adenoviral DNA was isolated from purified Ad-dl327 viruses by Hirt extraction, cleaved with Clal, and the large (approximately 35 kb) fragment was isolated by agarose gel electrophoresis and purified. The Clal fragment was used as the backbone for all first generation adenoviral vectors, and the vectors derived from it are known as Avl.

Five micrograms of linearized plasmid DNA (pAvS6n-LacZ) and 2.5 μg of the large Clal fragment of Ad-dl327 then were mixed and co-transfected into a dish of 293 cells by the calcium phosphate precipitation method. After 16 hours, the cells were overlaid with a 1:1 mixture of 2% Sea Plague agar and 2x medium and incubated in a humidified, 37°C, 5% C0₂/air environment until plaques appeared (approximately one to two weeks) . Plaques were selected and intracellular vector was released into the medium by three cycles of freezing and thawing. The lysate was cleared of cellular debris by centrifugation. The plaque (in 300 μl) was used for a first round of infection of 293 cells, vector release, and clarification as follows: One 35 mm dish of 293 cells was infected with 100 μl of plaque lysate plus 400 μl of IMEM-2 (IMEM plus 2% FBS, 2mM glutamine ⁽Bio Whittaker 046764)) plus 1.5 ml of IMEM-10 ⁽Improved minimal essential medium (Eagle's) with 2x glutamine plus 10% vol ./vol . fetal bovine serum) plus 2mM supplemental glutamine (Bio Whittaker 08063A) and incubated at 37°C for approximately three days until the cytopathic effect, a rounded appearance and "grapelike" clusters, was observed. Cells and supernatant were collected and designated as CVL-A. AvlLacZ4 vector (a schematic of the construction of which, is shown in Figure 6) was released by three cycles of freezing and thawing of the CVL-A. Then, a 60 mm dish of 293 cells was infected with 0.5 ml of the CVL-A plus 3 ml of IMEM-10 and incubated for approximately three days as above. Cells and supernatant from this infection then were processed by three freeze/thaw cycles in the same manner. AvlLacZ4 also is described in Yei, et al . , Human Gene Therapy, Vol. 5, pgs. 731-744 (1994); Trapnell, Advanced Drug Delivery Reviews, Vol. 12, pgs. 185-199 (1993) , and Smith, et al . , Nature Genetics, Vol. 5, pgs. 397-402 (December 1993) , which are incorporated herein by reference.

The resultant viral stock was titered by plaque assay on 293 cells using a standard protocol involving a 1.5 hour adsorption period in DMEM/2% FBS, followed by washout and agar overlay of the cell monolayer. (Graham, et al . , Virology, Vol. 52, pgs. 456-467 (1973)) . The absence of wild-type virus was checked by polymerase chain reaction assays of the stock using primers amplifying a 337 bp fragment of the El gene. The stock was negative for wild- type adenovirus using this assay.

The virus stock then was frozen at -80°C and stored until used. The virus stock had a titer of 1.5x10" pfu/ml. AvlTKl was constructed by inserting an HSV-TK fragment under the control of the RSV promoter and the major late mRNA tripartite leader of pAvS6. Details of such construction are as follows: pAvS6 was linearized with EcoRV, and the ends of the plasmid were treated with calf intestinal alkaline phosphatase ⁽CIAP) to prevent self-ligation. The Herpes Simplex Virus thymidine kinase (TK) gene was obtained from pGlNaSvTKl. (Figure 7) . pGlNaSvTKl includes a 1,225 bp fragment that contains 56 bp of TK 5' untranslated region and the full-length TK open reading frame as described in PCT Application No. W095/06486, published March 9, 1995. pGlNaSvTKl (Figure 7) was digested with Clal and Bglll_. to remove the Herpes Simplex Virus thymidine kinase gene. The ends of the gene were blunted with Klenow fragment, and the TK gene was gel purified from the retroviral backbone. The linearized pAvS6 and the TK gene were ligated together to form pAvSδ.TKl. (Figure 7) The generation of AvlTKl is nearly identical to that described for the construction of AvlLacZ4, except that 5μg of supercoiled pAvS6.TKl and 7.5μg of Ad dl327 Clal fragment were used in place of pAvS6-nLacZ and 2.5μg of Ad dl327 Clal fragment. Putative positives were screened by slot-blotting with a TK probe and were confirmed by Southern analysis. Plaque purification, scale-up¹, and tests for wild-type virus are as described with respect to AvlLacZ4.

AvlAFPTKl contains the 4.9 b Hindlll-Hindlll fragment of the 5' flanking sequences of the alphafetoprotein gene instead of the RSV promoter and tripartite leader upstream of the HSV-TK gene. (Ives, et al. , Methods in Enzvmology LI- Purine and Pyrimidine Nucleotide Metabolism, pgs. 337-345, Hoffee, et al. , eds . , New York, Academic Press, 1978) . AvlAFPTKl was constructed as follows: pAvS6 was digested with Ndel and Clal. This digest released the tripartite leader (TPL) sequence fragment from the backbone of the adenoviral shuttle plasmid, pAvS . The pAvS fragment was gel purified. The following single-stranded DNA oligomers:

5^'TATG^AA^AAT<_TAG^ATGATCAGCTAGCTACGfAGACGTCGα^"GGCACTAGTTCGCGAAGATCTGTTAACGTCGACGATATCAT-.V and

5^{' C}GATG^ATΛTCGTCGACGTTAACAGATCTTCGCGAACTAGTGCCGGCGACGTCTACGTAGCTAGCTGATCATCTAGATTTTCAV were prepared using a DNA synthesizer. The oligomers were annealed to form a double-stranded multiple cloning site including the Ndel, Xbal, Bell, Nhel, SnaBI, Aatll, Nael, Spel, Nrul, Bglll, Hpal, Sail, EcoRV, and Clal sites. The Ndel and Clal overhangs resulted at the 5' and 3' ends, respectively. The pAvS fragment and the multiple cloning site then were ligated to form pAvS12 (Figure 8) . pAvS12 then was digested with Mlul and Ndel. The digest released the RSV promoter from the backbone of the adenoviral shuttle plasmid, pAv. The pAv fragment was gel purified.

Plasmid pDG7 (Chang, et al . , J. Virol. , Vol. 64, No. 1, pgs. 264-277 (January 1990)) contains a chloramphenicol transferase (CAT) gene controlled by a simian CMV promoter. Using pDG7 as a template and the following primers:

S--AATTTCCATATGGAGCrrCCTtCGACGTCCC)CAGGCAGAATtTG-3- which contains an Ndel site, and

5-ATTCCAACGςGTTGGCCAGG)TTCAATACTAtrσTATTGGCCpr-3- which contains an Mlul site, the simian CMV promoter was duplicated by PCR. The ends of the simian PCR fragment were digested with Mlul and Ndel.

This fragment then was ligated into Mlul/Ndel digested pAvS12 (i.e., the pAv fragment) to form pAvSCMV. (Figure 9) . pAvSCMV was cut with SnaBI, and the ends of the plasmid were treated with calf intestinal phosphatase. pSPTKl (described in PCT Application No. W095/06486) was digested with Bglll and Xhol to isolate the TK1 fragment (also described in PCT Application No. W095/06486) . The ends of this fragment were blunt ended with Klenow, and this fragment was ligated to SnaBI digested pAvSCMV to form pAvSlO.TKl. (Figure 10) .

The unique Spel site of pAvSlO.TKl (Figure 11) was removed by cleaving with Spel, filling in the ends with the large fragment of E. col i DNA polymerase (Klenow) , and relegating. The new plasmid, pAvS19.TKl (Figure 11) was confirmed to have the Spel site removed, as verified by attempting to digest the plasmid with Spel. pAF4.9/Htoxin (Figure 11) was employed to supply the alphafetoprotein promoter. pAF4.9/Htoxin is a construct similar to pAF4.9-CAT (Watanabe, et al . , J.Biol.Chem.. Vol. 262, pgs. 4812-4818 (April 5, 1987)) except that the chloramphenicol transferase gene is replaced with a diphtheria toxin gene. pAF4.9/HToxin was constructed as follows:

pBR-CAT was constructed by inserting the Hindlll-BamHI fragment obtained from pSVO-CAT (Gorman, et al . , (1982) Mol. Cell Biol. 2:1044) into pBR322 purchased from BRL, Gaithersburg, MD according to the method of Walker, et al. , (1983) Nature 306:557. This plasmid contains the CAT coding sequence and the SV40 polyadenylation signal but lacks the SV40 enhancer and early promoter elements. pAFl.O-CAT then was constructed by inserting the 980 bp sequence between -951 and +29 relative to the cap site of the human AFP gene into the Hindlll site at the 5' end of the CAT gene of pBR-CAT.

To construct pAF5.1-CAT containing the 5.1 kb of the AFP 5'- flanking sequence, pAFl.O-CAT was digested completely with PstI, partially with EcoRI and a 5.7 kb DNA sequence containing a 0.9kb EcoRI-Hindlll human AFP DNA fragment (-870 to +29) , the CAT gene and the pBR322 replication origin was recovered.

The diphtheria toxin (DT) gene, a known gene, codes for diphtheria toxin consisting of an A and B-chain. It is known that diphtheria toxin can kill eukaryotic cells by blocking protein synthesis. The B-chain is involved in the binding of the A-chain to a cell. The sequence of the A and B chain of the DT gene is published. (Greenfield, L., et al . , Proc. Natl. Acad. Sci. USA, 80, 6853-6857 (1983)) . A DNA fragment coding for a modified A-chain (HToxin) was used. This modified DNA fragment was made by using PCR synthesis. This DNA fragment (HToxin) encodes for an A-chain having Cys as the 66th amino acid and Arg as the 194th amino acid, instead of the natural A-chain having Tyr and Pro, respectively, as reported by Greenfield and others . This DNA fragment was provided with a Hind III restriction enzyme cleavage site at the N-terminal and a Hpal restriction enzyme cleavage site at the C-terminal, using synthetic primers containing the Hind III or the Hpal site, respectively. pSV2-CAT (Gorman, et al. , Mol . Cell. Biol., 2, 104

(1982)) was digested by Hind III and Hpal to remove the CAT gene, and the DNA fragment encoding the HToxin DNA was inserted into the plasmid to construct a recombinant plasmid pSV2/HToxin. pSV2/HToxin was digested by Hind III and BamHI to obtain a DNA fragment having 759 bp which included the HToxin gene. The 759 bp DNA fragment was inserted in between the Hind III and BamHI sites of the pGEM-7Z vector (Promega) , thus constructing pGEM7Z/HToxin. The plasmid pAF4.9/HToxin comprises the entire transcriptional regulatory region of the AFP gene, and the HToxin gene controlled by the transcriptional regulatory region.

The 4.9 kb 5'-flanking region of the AFP gene was obtained by partially digesting pAF5.1-CAT (Example 1) with . Hind III, electrophoresing the resultant fragments in a 0.4% agarose, then collecting the 4.9 kb DNA fragment. The collected fragment was inserted into the Hind III site of the above-mentioned pGEM7Z/HToxin, thus obtaining the desired plasmid, pAF4.9/HToxin.

An intermediate plasmid then was constructed by performing PCR between the 3' Hindlll site which is at the 3' end of the promoter of pAF4. /Htoxin and approximately 200 bp upstream of this Hindlll site including the Spel site. Both AsuII and AscI sites were included in the primer designed for

-21-

UBSTITUTE SHEET (RULE 26) the 5' site, and an Nhel site was added into the 3' primer that is slightly downstream of the Hindlll site. The primer including the AsuII and AscI sites, which was designed for the 5' site, had the following sequence:

5' -ATGAGAGGCGCGCCTTCGAAGCTATGCTGTTAATTATTGGCAAA-3 ' The 3' primer including the Nhel site had the following sequence:

5' -ATCAGAGCTAGCAAGCTTGqGATCCGGTGTTAT-3 ' Following PCR, the band obtained was digested with AscI and Nhel and ligated to pAvS19.TKl digested with AscI and Nhel. The newly formed plasmid, pAvS20.TKl (Figure 11) also was confirmed by standard restriction digestion. pAvS20.TK1, which contains only the most 5' 200 bp of the alphafetoprotein promoter then was digested with AsuII and Spel, purified, and religated with the rest of the alphafetoprotein promoter contained on a 4.7 kb AsuII/Spel fragment obta_ned from digestion and agarose gel purification of pAF4.9/Htoxin. The resulting plasmid, pAvS21.TKl (Figure 12) , was confirmed to have the full length alphafetoprotein promoter upstream of TK by restriction digests . The junctions and PCR portions of this plasmid also have been sequenced to completion and agree with the expected sequence. The construction of the adenoviral vector AvlAFPTKl is nearly identical to that described for the construction of AvlLacZ4 with the following exceptions. 5μg per cotransfection of pAvS21.TKl linearized by Kpnl and purified by agarose gel electrophoresis and ion exchange resin beads (Gene Clean II, Bio 101) was used in place of pAvS6-nLacZ. Putative positives were screened by slot blotting with a TK probe and confirmed by Southern analysis. Plaque purification, scale- up, and tests for wild-type virus is as that described for AvlLacZ4. The final virus preparation had a titer of approximately 2.7x10" pfu/ml and was free of wild-type contamination. Example 2 Exponentially growing cells of the cell lines HuH_{7 ;} SK-Hep-1; Hep3B; HepG2 hepatocellular carcinoma cell lines, and of the HeLa cell cell line, were seeded onto duplicates of 12-well tissue culture dishes at a concentration of 2x_l0⁵ cells/well. The next day, serial dilutions of different multiplicities of infection (MOD of AvlLacZ4 were added as follows: 2xl0⁸ (MOI = 1,000) ; 2xl0⁷ (MOI = 100) ; 2xl0⁶ (MOI = 10) , and 2 x 10⁵ (MOI = 1) plaque forming units (pfu) per well. The following day, the cells were stained with 5- bromo-4-chloro-3-indolyl-B-D-galactoside (X-gal) after fixation with 0.5% glutaraldehyde. The number of lacZ- positive and negative cells in three high-power fields from each well was recorded, and the percentage of positive cells is presented as the mean + standard deviation. The results are given in Table I below.

For all five cell lines, at an MOI of 1,000, at least 96% of the attached cells were transduced while the cytopathic effect of the virus was evident. Lower MOI's of the vector resulted in proportionally reduced transduction efficiencies. The efficiencies in SK-Hep-1 were lower at MOI's of 10 and 100 compared with those in the other three hepatocellular carcinoma cell lines. Example 3 HuH7 or Sk-Hep-1 hepatocellular carcinoma cells, or HeLa cells, were seeded onto T12.5 tissue culture flasks at a concentration of 8xl0⁵ cells/flask. The next day either (i) AvlAFPTKl was added at multiplicities of infection of 10, 100, 1,000, or 5,000; (ii) AvlTKl was added at multiplicities of infection of 1, 10, or 100; or (iii) AvlLacZ4 was added at a multiplicity of infection of 10. Sixteen hours after infection, the cells were harvested using a cell lifter. After washing two times with 2 ml of lysis buffer containing lOmM Tris HC1 (pH7.5), ImM DTT, ImM EDTA, and 20% glycerol, the cell pellet was suspended in 0.2 ml of lysis buffer containing 200 μg/ml of Pefabloc SC (Boehringer Mannheim, Indianapolis, Indiana) , 40 μg/ml of aprotinin (Boehringer Mannheim)., and 5 μg/ml leupeptin (Boehringer Mannheim) . The cell lysate was obtained by centrifugation after 5 times of freezing and thawing of the cells.

HSV-thymidine kinase activity was determined as described in Ives, et al. , 1978, with a slight modifica...on. A 50 μl sample of reaction mixture containing 50 mM Tris HCL (pH 8.0) , 10 mM MgCl₂, 10 mM ATP, 45 μM tritium labeled gancyclovir (1 μCi) (Moraved Biochemicals, Inc., California) , and 37.5 μg of cell lysate was incubated at 37°C for 1 hour. A 40 μl aliquot then was spotted onto a Whatman DEAE-82 filter, and washed three times with ethanol. The amounts of phosphorylated gancyclovir were determined by scintillation counting. All experiments were carried out in duplicate and the mean cpm was determined. The results are given in Table II below.

AvlAFPTKl 100 128 0 —

AvlAFPTKl 1, 000 361 15 35

AvlAFPTKl 5,000 1,757 43 —

AvlTKl 1 103 65 —

AvlTKl 10 849 110 —

AvlTKl 100 17,472 5,270 32,579

AvlLacZ4 10 0 0 --

At multiplicities of infection of 1,000 and 5,000, the transduced cells demonstrated a cytopathic effect by the virus. In the alphafetoprotein producing cell line HuH7, there appeared to be a relationship between the enzymatic activity and the multiplicity of infection of AvlAFPTKl. The activity in the non-alphafetoprotein producing cell line SK- Hep-1, however, was significantly lower even at an MOI of 5,000. The alphafetoprotein-negative non-hepatocellular carcinoma cell line HeLa also demonstrated low activity of the enzyme. Higher HSV thymidine kinase activity was found in all three cell lines which were infected with AvlTKl although the activity in SK-Hep-1 was low compared with that found for HuH7 and HeLa. The HSV thymidine kinase activity by AvlAFPTKl transduction into HuH7 was lower than that by AvlTKl transduction. Thus, tumor cell specific expression of the HSV-TK gene was demonstrated in hepatocellular carcinoma cells .

Example 4

The sensitivity to gancyclovir of infected cells was measured with a non-radioactive cell proliferation assay according to the manufacturer's protocol (Cell Titer 96™ AQueous Non-Radioactive Cell Proliferation Assay; Promega, Madison, Wisconsin) .

4xl0³ HuH7 or SK-Hep-1 cells were plated in triplicate wells of a 96 well plate. The next day, the cells were infected with AvlAFPTKl, AvlTKl, or AvlLacZ4 at multiplicities of infection of 0, 1, 10, 100, or 1,000. Sixteen hours after infection, increasing concentrations of gancyclovir (Syntex Laboratories, Palo Alto, California) were added in amounts of 1, 10, and 50 μg/ml. Culture medium containing gancyclovir was changed every day. Four days after gancyclovir treatment, assay reagents (Cell Titer 96™ AQueous Non-Radioactive Cell Proliferation Assay; Promega, Madison, Wisconsin) were added, and the absorbance at 490 nm was measured. The percentage survival of cells is given as a percentage of the absorbance found in gancyclovir-treated cells divided by that in the cells without gancyclovir treatment . (mean ± standard deviation) .

The results are shown in Figures 13 through 18. Cytopathic effect by the adenoviral vectors was clear when infected at high multiplicities of infection. HuH7 cells infected with either AvlTKl or AvlAFPTKl at an MOI of 1,000 were killed totally after 4 days in the absence of gancyclovir treatment. (Figures 13 and 15) . HuH7 cells infected with AvlAFPTKl exhibited gancyclovir sensitivity at as low a concentration of gancyclovir of 1 μg/ml at an MOI of 100. (Figure 13) . SK-Hep-1 cells infected with AvlAFPTKl did not show any gancyclovir sensitivity even at high concentrations of gancyclovir at an MOI of 100, although there was a slight decrease in % survival at 50 μg/ml when infected at an MOI of 1,000. (Figure 14) . Both cell lines infected with AvlTKl similarly became sensitive to gancyclovir. (Figures 15 and 16) . Infection with a control adenoviral vector, AvlLacZ4, did not demonstrate gancyclovir sensitivity in either cell line. (Figures 17 and 18) . The above experiments indicate that AvlAFPTKl mediated transfer of the HSV-TK gene resulted in gancyclovir killing of only alphafetoprotein producing cells. Example 5 Sixty adult (7 weeks old) athy ic nude mice (Harlan Sprague Dawley, Inc., Indianapolis, Indiana) were injected subcutaneously in the right flank with hepatocellular carcinoma cells. 30 mice were injected with 10⁷ HuH7 cells and 30 mice were injected with 5x10° SK-Hep-1 cells. The mice injected with each cell line were divided into 6 groups according to the treatment schedules: (i) AvlAFPTKl injection and gancyclovir treatment (5 mice) ; (ii) AvlAFPTKl injection without gancyclovir treatment (5 mice) ; (iii) AvlTKl injection with gancyclovir treatment (5 mice) ; (iv) AvlTKl injection without gancyclovir treatment (5 mice) ; (v) AvlLacZ4 injection with gancyclovir treatment (5 mice) ; (vi) injection of dialysis buffer used for adenovirus preparation with gancyclovir treatment (5 mice) . Five days after injection of HuH cells and 9 days after injection of SK-Hep-1 cells, lxlO⁹ pfu of adenoviral vectors in a total volume of 100 μl of dialysis buffer were injected directly into the growing tumor from three directions for two successive days. The needle was retracted over 10 seconds. The next day, gancyclovir was administered intraperitoneally at 100 mg/kg once daily for 10 days. The size of the tumor was measured twice weekly with calipers in three dimensions. The tumor size is given as the mean + standard deviation.

The results are shown in Figures 19 and 20. Tumor growth in each treatment group was similar in size when the adenoviral vectors were injected into each hepatocellular carcinoma model (HuH7-83.2 mm³ ± 3.5; SK-Hep-1-10.9 mm³ ± 0.3) . All ten HuH7 tumors which received either AvlAFPTKl or AvlTKl demonstrated complete regression after gancyclovir treatment, while all ten tumors without vector injection markedly increased their size even after gancyclovir treatment. Also, injection of a total of 2xl0⁹ pfu of AvlLacZ4 also inhibited tumor growth. The combination of HSV thymidine kinase expression with gancyclovir treatment clearly was necessary to obtain complete regression of the tumor as demonstrated in the tumors which were injected with either AvlAFPTKl or AvlTKl in the absence of gancyclovir administration.

There was no complete regression in SK-Hep-1 tumors which received AvlAFPTKl and gancyclovir treatments. All five SK-Hep-1 tumors, however, which received AvlTKl injection and gancyclovir administration showed complete regression similar to that of the HuH7 tumors. Thus, the combination of AvlTKl and gancyclovir could kill HuH7 and SK- Hep-1 tumors, whereas the combination of AvlAFPTKl and gancyclovir only was effective in killing the alphafetoprotein producing HuH7 tumors.

The disclosure of all patents, publications (including published patent applications) , database accession numbers, and depository accession numbers referenced in this specification are specifically incorporated herein by reference in their entirety to the same extent as if each such individual patent, publication, and database accession number, and depository accession number were specifically and individually indicated to be incorporated by reference.

It is to be understood, however, that the scope of the present invention is not to be limited to the specific embodiments described above. The invention may be practiced other than as particularly described and still be within the scope of the accompanying claims.

Claims

WHAT IS CLAIMED IS:

1. A method of treating hepatocellular carcinoma in a host, comprising: administering to said host a DNA virus vector including at least one DNA sequence encoding at least one agent capable of providing for the inhibition, prevention, or the destruction of the growth of the cells of said hepatocellular carcinoma, and a promoter specific for liver tumor cells controlling said at least one DNA sequence encoding said at least one agent, said DNA virus vector being administered in an amount effective to inhibit, prevent, or destroy the growth of the cells of said hepatocellular carcinoma.

2. The method of Claim 1 wherein said DNA virus vector is an adenoviral vector.

3. The method of Claim 1 wherein said agent is a negative selective marker, and said method further comprises administering to said host an interaction agent which interacts with the negative selective marker to prevent, inhibit, or destroy the growth of the hepatocellular carcinoma.

4. The method of Claim 3 wherein said negative selective marker is selected from the group consisting of Herpes Simplex Virus thymidine kinase; cytomegalovirus thymidine kinase; varicella-zoster virus thymidine kinase; . and cytosine deaminase.

5. The method of Claim 4 wherein said negative selective marker is selected from the group consisting of Herpes Simplex Virus thymidine kinase; cytomegalovirus thymidine kinase; and varicella-zoster virus thymidine kinase .

6. The method of Claim 5 wherein said interaction agent is selected from the group consisting of gancyclovir, acyclovir, and i-2-deoxy-2-fluoro-/3-D-arabinofuranosil-5- iodouracil .

7. The method of Claim 6 wherein said interaction agent is gancyclovir.

8. The method of Claim 6 wherein said interaction agent is administered in an amount of from about 5 mg/kg to about 15 mg/kg.

9. The method of Claim 4 wherein said negative selective marker is cytosine deaminase.

10. The method of Claim 9 wherein said interaction agent is 5-fluorocytosine.

11. The method of Claim 1 wherein said promoter specific for liver tumor cells is the alphafetoprotein promoter.

12. A DNA virus vector including (i) at least one DNA sequence encoding at least one agent capable of providing for the inhibition, prevention, or the destruction of the growth of tumor cells; and (b) a promoter specific for liver tumor cells controlling said at least one DNA sequence encoding said at least one agent.

13. The vector of Claim 12 wherein said DNA virus vector is an adenovirus vector.

14. The vector of Claim 12 wherein said at least one agent is a negative selective marker.

15. The vector of Claim 14 wherein said negative selective marker is selected from the group consisting of Herpes Simplex Virus thymidine kinase; cytomegalovirus ■ thymidine kinase; varicella-zoster virus thymidine kinase; and cytosine deaminase.

16. The vector of Claim 15 wherein said negative selective marker is selected from the group consisting of Herpes Simplex Virus thymidine kinase; cytomegalovirus thymidine kinase; and varicella-zoster virus thymidine kinase.

17. The vector of Claim 16 wherein said negative selective marker is Herpes Simplex Virus thymidine kinase.

18. The vector of Claim 12 wherein said promoter specific for liver tumor cells is the alphafetoprotein promoter.

19. A composition comprising: a lipid vesicle; and a genetic construct encapsulated within said lipid vesicle, said genetic construct including (a) at least one DNA sequence encoding at least one agent capable of providing for the inhibition, prevention, or destruction of the growth of tumor cells; and (b) a promoter specific for liver tumor cells controlling said at least one DNA sequence encoding said at least one agent .

20. The composition of Claim 19 wherein said genetic construct is a plasmid vector.