Recombinant adenoviral vectors and methods of its use

 

(57) Abstract:

The invention relates to a recombinant adenovirus expression vectors, characterized by partial or complete deletion of the DNA fragment of adenovirus encoding the protein IX, and containing the gene of a foreign protein, or a functional fragment or mutant form. The proposed pharmaceutical composition comprising a recombinant adenoviral expression vector, which contains the insertion of exogenous DNA that contains a gene that encodes a foreign protein, and the adenovirus DNA having a deletion starting at position from 357 to 360 and ending in position from 4020 to 4050. Mentioned pharmaceutical composition can be used in gene therapy to transform of hyperproliferative mammalian cell therapy of cancer, the inhibition of proliferation of tumor in animals, to reduce the proliferation of cancer cells. 7 C. and 13 C.p. f-crystals, 2 tab., 16 Il.

For the formation of recombinant adenoviruses applicable in gene therapy, it is necessary to use the cell line in which "TRANS" type synthesized products of viral genes E1 region, deletirovannykh the source of viruses. Currently available only cell Lin the nome of adenovirus type 5 (Aiello, 1979; Spector, 1983).

Adenoviral vectors are investigated at the moment for the purposes of gene therapy, usually have deletions of genes Ad2 or Ad5, located from a point located at a distance of 400 KB from the 5'-end of the viral genome to the point, spaced approximately 63.3 KB from the 5'-end, with the common deletion region E1 (2.9 KB). Thus, there is a limited region of homology of approximately 1 KB between the DNA sequences of the recombinant virus, Ad5 DNA in cell lines. This homology determines the area of potential recombination between viral and cellular adenoviral sequences. This recombination leads to the formation of virus phenotypic wild-type, weight-bearing region of Ad5 E1 from 293 cells. Apparently, it is this recombination event leads to the frequent detection of adenovirus wild-type recombinant virus. In addition, it was directly shown that such recombination is the cause of contamination by wild-type virus recombinant virus Ad2/CFTR-1, created on the basis of Ad2 (Rich et al., 1993).

Due to the high degree of homology of sequences in the subset of adenovirus type C, such recombination is more likely if to create a vector isprovided the problem of contamination by wild-type virus can be solved by the selection procedure, when contaminated batch of virus simply discarded. With increasing cultivation for purposes of gene therapy increases the likelihood of contamination of each batch of virus with wild-type virus and increase the difficulty of obtaining decontaminating of recombinant virus.

This year will be diagnosed with more than one million cases of primary cancer, and the number of deaths due to cancer will reach half a million (American Cancer Society, 1993). Mutations in p53 gene are the most frequent genetic damage associated with human cancers, they are found in 50-60% of human cancers (Hollstein et al.,1991: Bartek et al., 1991: Levine, 1993). The aim of gene therapy p53-deficient tumors is, for example, the introduction of normal functional copy of the gene p53 wild-type to restore the control of cell proliferation. P53 plays a key role in cell cycle, stopping the growth, so that could be the repair or apoptosis in response to DNA damage. Recently it was shown that p53 wild type is a necessary component of the apoptosis induced by irradiation or treatment with certain chemotherapeutic is pleasant, these tumors have become resistant to chemo - and radiotherapy due to the loss of p53 wild-type. "The delivery of functional p53 in these tumors with a high probability will make them susceptible to apoptosis, usually associated with DNA damage induced by radiation or chemotherapy.

One of the critical elements in successful therapy of human diseases using gene tumor suppressor is the ability to impact on a significant proportion of tumor cells. To this end, various tumor models widely used retroviral vectors. For example, for the treatment of liver tumors the use of retroviral vectors have been ineffective, because they were not able to achieve a high level of transfer of genes required for gene therapy in vivo (Huber, B. E. et al., 1991; M. Caruso et al., 1993).

To create more long-term source of production of virus researchers have attempted to overcome the problem of low frequency of gene transfer by direct injection into solid tumors packaging cells producing retroviral vectors (Caruso, M. et al., 1993; Ezzidine, Z. D. et al., 1991; Culver, K. W. et al., 1992). However, this approach proved to be unacceptable for the treatment of patients, because in response to the introduction of the s is their need for dividing cells for effective integration and recombinant expression of transferred gene (Hubner, C. E., 1991). Stable integration of the retroviral genome in an essential gene of the host cell may lead to the occurrence and inheritance of various pathologies.

Recombinant adenoviruses have significant advantages compared to retroviral vectors and other methods of gene transfer (see review Siegfried, 1993). Never been shown that adenoviruses can cause tumors in humans and quite safely used as live vaccines (Straus, 1984). Replication defective recombinant adenoviruses can be obtained by replacing the field E1 required for replication, a gene that is designed to move. Adenovirus does not integrate with the genome of human cells, and thereby greatly reducing the risk of insertional mutagenesis, possible when using retroviral and adeno-associated (AAV) vectors. The lack of stable integration improves safety and even due to the fact that the effect of the transferred gene is temporary, as extrachromosomal DNA will be lost as dividing normal cells. Stable recombinant adenovirus with a high titer can be obtained in quantities not achievable in the case of retroviral or AAV vectors that conductively gene transfer in vivo in a variety of tumor cells and tissues. For example, it was shown that gene transfer using adenovirus is essential for the gene therapy of diseases such as testoviron (Rosenfeld et al., 1992; Rich et al., 1993) and deficiency of alpha-1-antitrypsin (Lemarchand et al.,1992). Although currently used and alternative methods of gene transfer, such as cationic complexes of liposomes/DNA, none of these methods is not yet as effective as gene transfer using adenovirus.

As in the case of defective in p53 tumors, the goal of gene therapy for other tumors is regaining control over cell proliferation. At deficiency of p53 introduction of a functional gene regains control of the cell cycle and makes it possible cell death by apoptosis under the action of therapeutic drugs. Similarly, hemotherapy equally can be based on the manipulation of other genes tumor suppressor, which can be used independently or in combination with therapeutic drugs for the control of the cell cycle of tumor cells and/or inducing cell death. In addition, genes that do not encode proteins - regulators of the cell cycle, but directly cause cell death, such as suicide" hedorah for cell cycle arrest in tumor cells.

Regardless of which gene is used to regain control of the cell cycle, theoretical background and the practical importance of this approach remain unchanged. Namely, it is necessary to obtain a high efficiency of gene transfer for the expression of therapeutic quantities of the recombinant product. For successful gene therapy approach is important the right choice of vector, providing a high efficiency of gene transfer with minimal risk for the patient.

Thus, there is a need in the vectors and methods, providing high efficiency of gene transfer and high level expression of proteins that would be safe enough for gene therapy procedures. The present invention meets these objectives and, in addition, provides a number of additional advantages.

In Fig. 1 presents claimed in the present invention is an adenoviral vector. The Assembly of the construct was produced as shown in Fig. 1. The resulting virus has a 5'-end deletion of the adenoviral sequences extending from nucleotides 356 to nucleotide 4020 and eliminating genes E1a and E1b, and all coding sequences of the protein IX, leaving intactos the desired gene.

In Fig. 2 shows the amino acid sequence p110RB.

In Fig. 3 shows the DNA sequence that encodes a protein suppressor retinoblastoma.

In Fig. 4 schematically presents the recombinant constructs of P53/adenovirus claimed in the present invention. P53-recombinants based on Ad 5, which region E1 (nucleotides 360-3325) was replaced by a full size (1.4 KB) cDNA of p53. While the expression of p53 was going promoter Ad 2 MLP (A/M/53) or the promoter of the human cytomegalovirus (CMV) (A/C/53), followed by a three-membered leader cDNA Ad 2. The reference virus A/M had the same deletion of the Ad genome 5, as the virus A/M/53, but not had a 1.4 KB insert cDNA of p53. The remaining sequence E1b (705 nucleotides) was deleterule to obtain constructs A/M/N/53 and A/C/N/53 with a deletion of the protein IX. These constructs also had a 1.9 KB Xba 1-a deletion in region E3 of adenovirus type 5.

Fig. 5A and 5B illustrate the expression of p53 protein in tumor cells, virus-infected A/M/53 and A/C/53. Fig. 5A: cells Saos-2 (osteosarcoma) were infected with the indicated multiplicity of infection with a virus (A/M/53 or virus A/C/53 and analyzed 24 hours after infection. Antibody pAb 1801 p53 used for painting immunodeficiency, the protein samples extracts of SW480 cells, which Express mutant p53 in large quantities. "About" under "A/C/53" means pseudoinfection, and this track contains lysate untreated cells Saos-2. Fig. 5B: cell hepatocellular carcinoma Hep B3 infected with a virus (A/M/53 or virus A/C/53 with the indicated multiplicity of infection and analyzed as described in section "A". The arrow indicates the position of the p53 protein.

In Fig. 6A-6C shows p53-dependent change in cell morphology Saos-2. Subconfluent cells Saos-2 (1 of 105cells/10 cm Cup) were left uninfected (A), infected with a multiplicity of 50 reference virus A/M (B) or a virus (A/C/53 (C). Cells were photographed after 72 hours after infection.

In Fig. 7 shows p53-dependent inhibition of DNA synthesis in tumor human cell lines infected with virus A/M/N/53 and A/C/N/53. Cell nine different tumor lines infected or control adenovirus A/M (-x-x-), or expressing A p53/M/N/53 (- - -) or A/C/N/53 (-Oh-) with increasing multiplicity of infection, as shown in the figure. For each cell line shows the type of tumor and the status of p53 (wt - wild type; null - protein is not expressed; mut - mutant protein is expressed). After 72 hours after infection relative dose (mean +/- standard deviation) and presented as percentage of control environment, depending on the multiplicity of infection. H69 cells were only tested virus A/M and A/M/N/53.

In Fig. 8 presents tumorogenic cells Saos-2 infected p53, naked mice. Cells Saos-2 infected or control virus (A/M or p53-recombinant A/M/N/53 with a multiplicity of infection of 30. Treated cells were injected subcutaneously in the flank of naked mice, and twice a week for 8 weeks to determine the size of the tumors (as described in Experiment II). The results were expressed as the dependence of the size of the tumor on days that have elapsed since implantation of tumor cells as control A/M - infected (-x-x-) cells, and A/M/M/53 - infected cells (- - -). Bounds on the errors reflect the average size of tumors (+/- standard deviation) for each group of 4 animals for each time point.

Fig. 9 illustrates the expression of rAd/p53 RNA in tumors. Naked mice was subcutaneously injected cells H69 (SCLC) and for 32 days were allowed to develop tumors, which by this time had reached a size of about 25-50 mm3. Then randomly selected mice were administered peritumorally 109plaque-forming units or control virus (A/C/betagan or virus A/C/53. On the 2nd and 7th days after injection, tumors were cut from each tumour sample was allocated the poly is plification was carried out for 30 cycles at 94oC 1 min, 55oC 1.5 min, 72oC 2 min, and final extension was 10 min at 72oC. Used thermocycler Omnigen (Hybaid). For PCR used the following primers: 5 trecchina leader cDNA (5'- CGCCACCGAGGGACCTGAGCGAGTC-3') and 3'p53 primer (5'- TTCTGGGAAGGGACAGAAGA-3'). On lines 1, 2, 4, and 5 are samples from treated p53 tumors obtained, as shown, on the 2nd or 7th days. On lines 3 and 8 were drawn samples from tumors treated with beta-Gal. Lines 7, 8 and 9 are respective repetitions of lines 4, 5 and 6, but the amplification was performed with primers actin to ensure equal loading. On line 10 shows the positive control with plasmid containing triclinic/p53.

In Fig. 10A and 10B shows the inhibition of tumors in vivo and increase survival time under action A/M/N/53. Naked mice was subcutaneously injected cells H69 (SCLC) and within 2 weeks was given the opportunity to develop tumors. Twice a week the mice peritumoral introduced or simply buffer (---), or a control adenovirus A/M (-x-x-), or a virus (A/M/N/53 (- - -) (the dose of both viruses was 2109the battle for injection, a total of 8 doses). The tumor size was determined twice a week and the volume of the tumors was assessed as described in Experiment N II. A) the size of the tumor is H69. Bounds on the errors reflect the average size of tumors (+/- standard deviation) for each group of 5 animals. Arrows indicate the days of the introduction of the virus. B) the share of the surviving mice for each group depending on the time elapsed after the introduction of H69 cells treated only with buffer (---), control virus A/M (... ... ...) or a virus (A/M/N/53 (_).

In Fig. 11A - 11C shows a map of recombinant plasmid constructs. Construction of plasmids were performed as described below. Bold lines in the constructs identified genes of interest, and bold the indicated restriction sites that are used to join the fragments to obtain plasmids, as indicated by the arrows. In Fig. 11A shows the construction of plasmids pACNTK by sublimirovanny gene HSV-TK in polylinker vector cloning, followed by separation TK gene with the desired ends for cloning into the vector pACM. The pACN vector contains the adenovirus sequence essential for recombination in vivo, leading to the formation of recombinant adenovirus (see Fig. 12). In Fig. 11B shows the construction of plasmids pAANTK starting with PCR amplification of fragments of the coding enhancer (AFP-E) and the promoter (AFP-P) gene alpha-flop the EP AFP are "above" gene HSV-TK, followed by adenovirus (Ad2) sequences required for recombination in vivo, leading to the formation of recombinant adenovirus. In Fig. 11C shows the construction of plasmids pAANCAT, beginning with the selection of commercially available plasmids gene chloramphenicolchloramphenicol (CAT) followed by sublimemovies it in plasmid pAAN (see above) to give the final plasmid pAANCAT, in which transcription of the CAT gene, surrounded by adenoviral sequences, is directed by the promoter/enhancer AFP.

In Fig. 12 shows a schematic map of recombinant adenoviruses ACNTK, AANTK and AANCAT. To obtain the recombinant adenoviruses of the plasmids shown in Fig. 11, 4 pieces (20 µg) of each of the plasmids pACNTK, pAANTK or pAANCAT were linearized with the restriction enzyme EcoRI and cotransfection 1 part (5 µg) large fragment restricciones Clal recombinant adenovirus (rACbeta-gal) containing a deletion region E3 (Wills et al., 1994). The resulting viruses nucleotides 360-4021 virus Ad5 or replaced by the CMV promoter and trinomial leader cDNA (TPL) or promoter/enhancer AFP directing the expression of a gene of HSV-1 TK gene CAT. The obtained recombinant adenoviruses are marked accordingly ACNTK, A adenoviral vectors. Two million cells (2 of 106the specified lines were infected with a multiplicity of infection of 30 or 100 recombinant adenovirus AANCAT or left uninfected (UN). Cells Hep G2 and Hep 3B expressed AFP, while the remaining cells are not expressed. After three days cells were collected, protein concentration in cell lysates was levelled and determined the activity of CAT, as described below in the Methods section. An equal number of uninfected cells served as a control background CAT activity, despite the fact that14C-chloramphenicol (only14C) and extract stable cell lines B21, expressing CAT, served as negative and positive controls, respectively. Specify the percent conversion of acetyl-CoA, it is clear that the CAT expression is limited to cells expressing AFP.

In Fig. 14 shows the effect of processing TK/GCV cells hepatocellular lines and the dependence of this effect on the specificity of the promoter. Cell lines Hep-G2 (AFP-positive) and HLF (AFP-negative) were infected during the night one of the following viruses: ACNTK [- -], AANTK [- -] or control ACN [--] with a multiplicity of infection equal to 30, and then was treated with a single dose of ganciclovir in the specified concentrations. The proliferation of clamidia in cellular nucleic acids were measured 72 hours after infection (TopCount, Packard) and expressed in percent (mean +/- mean deviation) relative to the untreated control. The results indicate non-selective dose-dependent suppression of cell proliferation under the influence of the construct with the CMV-promoter, while the TK gene under the control of the AFP-promoter selectively inhibits cells Hep-G2.

Fig. 15 illustrates the cytotoxicity ACNTK in combination with ganciclovir for cells of hepatocellular carcinoma (HCC). The HLF cells were infected with a multiplicity of infection of 30 virus ACNTK [--] or a control virus ACN [--], and then was treated with ganciclovir in the dose. After 72 hours after treatment with acyclovira colorimetrically determined the amount of lactate dehydrogenase (LDH) released into the cell supernatant. Shows a plot of the amount of LDH (mean +/- standard error) depending on the concentration of ganciclovir treated for two virus groups.

In Fig. 16A and 16B shows the effect ACNTK in combination with ganciclovir formed on hepatocellular cancer (HCC) in naked mice. Female Nude mice subcutaneously in the flank was injected ten million (1107cells, Hep 3B and in the course of 27 days was allowed to form tumors. Then the mice intratumoral lane or the ez day, all three doses (indicated by arrows). Injections of ganciclovir (100 mg/kg, administered intraperitoneally) was started 24 hours after the first injection of the virus and continued for 10 days.

In Fig. 6A shows a plot of the size of the tumors in the case of each virus on the number of days after infection (mean +/- the average error). In Fig. 6B shows a graph of the dependence of the mean body weight for each group of animals treated with virus, depending on the number of days elapsed from the moment of infection.

To reduce the frequency of contamination with wild-type adenovirus is desirable to reduce the ability of adenovirus or cell lines to recombination. For example, the adenovirus from the group with low homology with viruses of group C, can be used to construct recombinant viruses with a slight propensity for recombination with Ad5 sequences in 293 cells. However, the alternative, reduce the frequency of recombination between viral and cellular sequences can be achieved by increasing the size of the deletions in the recombinant virus and, therefore, reduce the total length of the sequence between him and AdS-genes 293 cells.

The adenovirus gene protein IX encodes a minor component of the outer adenoviral capsid, which stabilizes devyatisilnyi hexane comprising mainly viral capsid (Stewart, 1993). Studies of deletion mutants of adenovirus originally gave reason to believe protein IX is not a necessary component of adenovirus, although his absence was associated with increased compared with wild-type virus by thermolability (Colby and Shenk, 1981). Recently it was discovered that protein IX is required for full-sized packaging of viral DNA into the capsid, and that in the absence of this protein as a recombinant viruses can reproduce only those that contain the genome of at least 1 KB smaller than wild-type virus (Ghosh-Chooudhury et al., 1987). Taking into account these constraints deletion of the protein IX was not considered when constructing adenoviral vectors.

This application provides links to standard textbooks of molecular biology, which contain definitions and how to perform the basic techniques used in the present invention. See, for example, Sambrook et al., (1989) and cited in this book references. This book and the other cited publications included in the text of this pisaniello the use of recombinant adenoviruses, having deletions in the gene of the protein IX, which leads to reducing the risk of contamination by wild-type virus when receiving viral drugs for diagnostic and therapeutic purposes, such as gene therapy. The term "recombinant" means the viral progeny, formed as a result of genetic manipulations. These deletions may remove additional 500-700 base pairs of DNA sequences normally present in conventional E1-deletirovannykh viruses (possible smaller, less desirable deletions of parts of the gene p1X, which are also included in the scope of the present invention) and is available for recombination with sequences Ad 5, integrated into the genome of 293 cells. Recombinant adenoviruses based on any representative group C, serotype 1, 2, 5 and 6 included in the scope of the present invention. This invention also protected recombinant virus-based hybrid Ad2/Ad5, which provides the expression of p53 cDNA person under the main control of the late promoter of adenovirus type 2. This construct was assembled, as shown in Fig. 1. The resulting virus is 5'-deletion of the adenoviral sequences 357 th 4020-th nucleotide and deleted genes E1a and E1b, as well as all encoding members who allows to terminate the transcription of any desired gene. Alternatively, the deletion can be increased by 30-40 base pairs that do not affect neighboring gene protein IVa2, although in this case, you want exogenous polyadenylation signal for termination of transcription of the genes introduced into the recombinant virus. The original virus with this deletion is easily propagated in 293 cells without signs of contamination by wild-type virus and directs strong expression of p53 with transcriptional block entered in the site of the deletion.

The capacity of the recombinant virus having the above-described deletion of the gene of the protein IX is about 2.6 kilobase. This is sufficient for most genes, including p53 cDNA. The capacity of the vectors can be improved by the introduction of adenovirus "skeleton" other deletions, such as deletions in early regions 3 or 4 (see Graham and Prevec, 1991). For example, can be used adenoviral a "skeleton" with the deletion of 1.9 KB not necessary sequences in early region 3. With this additional deletion of the capacity of the vector increases approximately up to 4.5 kilobase, which is enough for most large cDNA, including cDNA suppressor gene retinoblastoma.

The present invention describes a recombinant adenoviral vector, characterized by the complete or partial UNT, or mutant. These vectors are applicable for safe recombinant obtain diagnostic and therapeutic polypeptides and proteins, and, more importantly, for the introduction of genes for gene therapy. So, for example, presented in this invention is an adenoviral vector may contain the foreign gene that directs the expression of a protein that participates in cell cycle regulation, such as p53, Rb, or mitosis, or protein, inducing cell death, such as encoded normal "suicide" gene timedancing. (The latter should be used in combination with metabolite timedancing to achieve the effect). In the present invention, the vectors may be used any of the expression cassettes. By "expression cassette" refers to a DNA molecule comprising a promoter/enhancer of transcription, such as the promoter/enhancer of cytomegalovirus (CMV), a foreign gene, and, as described below in some cases, the polyadenylation signal. The term "foreign gene" denotes a DNA molecule, is not presented in the correct orientation and in the correct position in the genomic DNA of wild-type adenovirus. The foreign gene may be a DNA molecule size to 4.5 kilobase. Vector exp is Chadasha cells masters, in other words, the protein or polypeptide encoded by this DNA is synthesized in these cells. Recombinant adenoviral expression vector may contain a portion of the gene encoding adenoviral protein IX, despite the fact that biologically active protein IX or its fragment is not produced. An example of such a vector is an expression vector, restriction map of which is shown in Fig. 1 or 4.

In the claimed in the present invention the vector may also be used inducible promoters. These promoters initiate transcription only in the presence of additional molecules. Examples of inducible promoters are the promoters of the genes beta-interferon, heat shock genes, gene metallothionein, as well as the promoters of genes expressed under the action of steroid hormones. Tissue-specific expression adequately studied, and tissue-specific and inducible promoters are well known in the prior art. These genes are used to regulate expression of a foreign gene after transfer into the target cell.

In the scope of the present invention also includes recombinant adenoviral expression vector, similar to that described above, but with less prot is orogeny from the point apart 3500 base pairs from the 5' end of the viral genome to the point, separated by about 4000 base pairs from the 5'-end. In another specific embodiment of the invention the recombinant adenoviral expression vector may also have additional deletion of non-essential DNA sequences of the adenovirus early region 3 and/or 4 and/or a deletion of DNA sequences of adenovirus, denoted as E1a and E1b.

In this case, the foreign gene may be a DNA molecule size to 4.6 kilobase. In another embodiment of the invention, the vector has a deletion in size to forty nucleotides located 3'-end in relation to the deletion of the E1a and E1b and p1X, as well as in the composition of the vector included alien DNA molecule encoding a signal predefiniowane located so against alien gene to regulate its expression.

According to the purposes of the present invention the recombinant adenoviral vector can be obtained from the group of wild-type adenovirus, serotype 1, 2, 5, or 6.

In one of the embodiments of the invention the recombinant adenoviral expression vector has as a foreign gene is a gene that encodes a functional protein suppressor of tumor or it is genes tumor suppressor, which encode suppressors of tumors, which in turn effectively inhibit the transformation of normal cells into tumor. Functional genes can include, for example, the normal genes of the wild type and modified normal genes that retain their ability to encode efficient suppressors of tumors, as well as other anticancer genes, such as coding suicide protein or toxin.

Similarly, the term "non-functional" is used in this context as a synonym of the term "inactivated". Non-functional or defective genes can occur as a result of various events, including, for example, point mutations, deletions, methylation, and other phenomena are well known from the prior art.

In the context of this invention under "active fragment" of a gene refers to smaller areas of the gene, which retain the ability to encode proteins with anti-tumour activity. P56 RB, described in more detail below, is one example of an active fragment of functional suppressor gene tumor. Modification of genes tumor suppressor, such as additions, deletions or substitutions, are also applicable in relation to their active fragments porom suppressor gene tumor is the retinoblastoma gene (RB). The complete nucleotide sequence of the RB cDNA and predicted amino acid sequence of the protein RB (marked p110RB) were published by Lee et al., (1987) and shown in Fig. 3. Also useful for the expression of retinoblastoma protein suppressor of tumor is a DNA molecule encoding the amino acid sequence shown in Fig. 2, or having the DNA sequence shown in Fig. 3. A subset of p110RB, p56RB, may also be useful. The sequence of p56RB published by Huang et al., (1991). In the present invention, the vectors can be used more genes tumor suppressor encoding the corresponding proteins. For illustration, you can name some of them: protein p16 (Kamb et al.,1994), p21 protein, protein WT1 tumor Wilma, mitosis, h-NUC or protein DCC carcinoma of the rectum. Mitosis described X. Zhu and W-H Lee in the application on invention USA N 08/141,239, filed October 22, 1993, and further partial continuation of said application by the same authors, legal statement N P-CJ 1191, issued October 24, 1994, Both documents cited in this application as references. Similarly, h-NUC described W-H Lee, and P. L. Chen in the application on invention USA N 08/170,586, filed December 20, 1993 and included in the EP amino acids, connected by peptide bonds in a specific sequence. The term "amino acid" refers to a D or L stereoisomeric forms of amino acids, unless otherwise indicated. In the scope of the present invention also includes equivalent proteins or equivalent peptides having biological activity of the purified protein suppressor of wild-type tumors. "Equivalent proteins" and "equivalent polypeptide" refers to compounds that differ from linear sequences of natural proteins or polypeptides, but which have amino acid substitutions that do not change their biological activity. These equivalents may differ from the original sequence by replacing one or more amino acids of similar amino acids, for example, similar to charged amino acids, or by replacement or modification of side chains or functional groups.

The definition of functional protein suppressor of tumor extends to any protein whose presence reduces tumorigenicity, malignancy or hyperproliferative phenotype of the host cell. According to this definition examples of genes are tumor suppressors include, but are not limited to the list) p110RB, p56RB, mitosis, h-NUC and p5 and is synonymous with neoplastic growth. "Malignancy" refers to the ability tumorogenic cells to metastasize and creating a threat to the life of the host organism. "Hyperproliferative phenotype" characterized by growth and division of cells, which occur at a rate greater than normal for cells of this type. The term "neoplastic" refers to cells that have lost functional endogenous protein suppressor of tumor, or inability of the cells to Express the endogenous nucleic acid encoding a functional protein is a tumor suppressor.

The example stated in the present invention the vector is a recombinant adenoviral expression vector containing the foreign gene is a gene that encodes p53 protein or its active fragment. The coding sequence of the p53 gene are shown in Table 1.

Any of the vectors described here the expression is applicable as a means of diagnosis or therapy. Vectors can be used for screening multiple genes tumor suppressor in respect of their application for gene therapy. For example, suspected of neoplasticity cells can be obtained from the patient or the animal. Then, in the corresponding conditions the present invention and carrying one functional suppressor genes. Does the introduction of this gene the reversion of malignant phenotype can be determined by colony in soft agar or the introduction of treated cells naked mice.

If malignant phenotype revertive, this gene may be regarded as a positive candidate for successful gene therapy to the patient or animal. When the pharmaceutical use of such a gene can be used in combination with one or more pharmaceutically acceptable carriers. These carriers are well known in the art and include aqueous solutions, as buffered saline or other solvents - glycole, glycerin, vegetable oils (e.g. olive oil) or organic esters suitable for injection.

Pharmaceutically acceptable carriers can be used for delivery of soluble compositions in a cell in vitro or for administration to a subject in vivo. Pharmaceutically acceptable carriers can contain physiologically acceptable substance which, for example, stabilizes the composition or reduces or increases the absorption agent. Such physiologically acceptable compounds can include, for example, carbohydrates such as glucose, shularni proteins or other stabilizers. Other physiologically acceptable compounds can be a moisturizing agents, emulsifiers, dispersing agents or preservatives are needed to prevent the growth of microorganisms. There are numerous preservatives, such as phenol and ascorbic acid. Experienced researcher understands that the choice of pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, how the introduction of the polypeptide and specific physico-chemical characteristics of the individual polypeptide. Such physiologically acceptable substances, as monostearate aluminum or gelatin, are particularly useful inhibiting agents that prolong the absorption introduced to the subject the pharmaceutical composition. Other examples of carriers, stabilizers and adjuvants can be found in the book Martin, Remington's Pharm. Sci. , 15th Ed. (Mack Publ.Co., Easton, 1975). The pharmaceutical compositions can be optionally incorporated into liposomes, microspheres or other polymer matrix (Gregoriages, Liposome Technology, Vol.l (CRC Press, Boca Raton, Florida, 1984). Liposomes consisting of phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolisable carriers that are relatively simple the composition, upominuemie in this invention, in combination with one or more of the above pharmaceutically acceptable carriers. Song data can be used in therapeutic or prophylactic purposes. They can be put into contact with the cells of the host in vivo, ex vivo or in vitro in effective amounts. In vitro and ex vivo means making contact with the cells of the host, as described below. In the case of in vivo methods of administration of the pharmaceutical preparation containing described in the present invention the vector is well known in the art and include, but are not limited to these) oral introduction, intratumoral, intravenous, intramuscular or intraperitoneal. The introduction can be constant or intermittent and may change depending on the condition of the patient being treated, and the nature of the disease, just as it happens in the case of other therapeutic compositions (Landmann et al., 1992; Aulitzky et al. , 1991; Lantz et al., 1990; Supersaxo et al.,1988; Demetri et al., 1989; LeMaistre et al., 1991).

In addition, the scope of the present invention included a transformed prokaryotic and eukaryotic cells - hosts such as mammalian cells or cells of other animals, in which wedensday (but are not limited to these) such bacterial cells, as E. coli cells. Methods of transformation of the host cells, the retroviral vectors are well known (see Sambrook et al., 1989) and include (but are not limited to these) transfection, electroporation and microinjection.

In the context of this application, the term "animal" is considered synonymous with the term "mammal" means (but is not limited to these animals) cow, pig, cat, monkey, dog, horse, mouse, rat or human. In addition, the cells of the host may include (but is not limited to these) any neoplastic or tumor cells, such as osteosarcoma, carcinoma of the ovary, breast cancer, melanoma, hepatocarcinoma, lung cancer, brain cancer, colon cancer, hematopoietic cells, prostate cancer, cervical carcinoma, retinoblastoma, carcinoma of esophagus, bladder cancer, neuroblastoma, or a cancer of the kidneys.

Furthermore, any cell line eukaryotes, is able to Express the E1a and E1b or E1a, E1b and p1X, is a suitable host for this vector. In one of the embodiments of the present invention, the host cells used eukaryotic cells 293, available from Amerikanskii cell culture collection (12301 Parklawn Drive, Rockville, Maryland, U. S. A. 20231).

therapy. When pharmaceutical use, they can be combined with various pharmaceutically acceptable carriers. Convenient pharmaceutically acceptable carriers well known in the art and, for example, some of them described above. Compositions in effective amounts can be used therapeutically or prophylactically, as described in more detail below.

The present invention also relates to a method of transformation of the host cell. This method consists in providing contact host cell under appropriate conditions, i.e., prokaryotic or eukaryotic host cell, with one of the expression vectors described in this application. Transformed according to the method of cell owners also included in the scope of the present invention. The contacting may be achieved in vitro, in vivo or ex vivo using methods known from the prior art (Sambrook et al., 1989), while the expression vectors used in effective quantities. In this application also includes a method of obtaining a recombinant protein or polypeptide when growing the transformed host cell under appropriate conditions conducive to transcription and translation of the rst is capitalship, yeast, insect or bacterial cells, are widely known and include, for example, described in Sambrook et al., supra. Translated alien protein can then be selected by conventional methods such as purification on columns or purification using antibodies to this protein. Isolated protein or polypeptide are also included in the scope of the present invention. With respect to protein, the terms "purified" or "isolated" means that the protein is substantially free from the original protein or nucleic acid, usually associated with the protein or polypeptide in its natural environment (cage master).

The present invention also relates to an animal bearing the introduced expression vectors and transformed cell hosts. Such transgenic animals are produced by methods well known in the prior art, for example as described in U.S. Patent N 5,175,384, or conventional methods of therapy ex vivo, as described Culver et al., 1991.

As described in more detail below, recombinant adenoviruses expressing tumor suppressor is p53 wild type, can effectively inhibit the synthesis of DNA and supressive growth of a wide spectrum of human tumor cells, including those that have it, such as p53, developed in vivo tumors without the need for direct injection into the tumor or pre-treatment of cancer cells ex vivo. Expressed p53 is functional and effectively suppresses tumor growth in vivo and significantly increases survival time, as shown in model naked mice inoculated with cells of lung cancer person.

Thus, as claimed in the present invention, the vectors are particularly suitable for gene therapy. Accordingly, the scope of the present invention is included and the methods of gene therapy using these vectors. Vector cleaned and then an effective amount of this vector is administered to a subject in vivo or ex vivo. Methods of gene therapy are well known in the prior art. See, for example, Larrik, J. W. and Burck, K. L. (1991) and Kreigler, M. (1990). "Subject" means any animal, mammal (cow, pig, cat, dog, horse, mouse, rat or human. Despite the fact that included in the vector queerty gene encodes a protein suppressor of tumor or other antitumor protein, this vector is applicable for the treatment of the subject and reduce the final number of hyperproliferative cells to suppress the proliferation of tumor cells or facilitate specific Specifica: hyperplasia of the thyroid gland - the Grave's disease, psoriasis, benign prostatic hypertrophy, syndrome Li-o Fraumeni, including breast cancer, sarcomas and other neoplasms, bladder cancer, colon cancer, lung cancer, various leukemias and lymphomas. Examples of non-pathological hyperproliferative cells are epithelial cells of the ducts of the mammary gland during lactation and cells associated with wound healing. Pathologic hyperproliferative cells are characterized by loss of contact inhibition and reduced capacity for selective attachment that reflects changes in the surface properties of cells and disrupting intercellular interactions. These violations include the stimulation of the division and the ability to secrete proteolytic enzymes.

In addition, in the present invention included the method of exhaustion of the corresponding sample patologicheskih hyperproliferative mammalian cell, which containerful hematopoietic precursors in the process of bone marrow transplantation. This is achieved by introducing a gene suppressing tumors in wild-type cells of the corresponding sample (obtained from autologous peripheral blood or bone marrow) group is n cell drug obtained from the patient, i.e., a mixed population of cells containing both phenotypically normal and pathological cells. The term "introduction" includes (but is not limited to the following) introduction to cell or intravenous injection, direct introduction into the tumor, intratumoral introduction, intraperitoneal, aerosol introduction into the lungs or local application. This procedure can be combined with the use of pharmaceutically acceptable carrier as described above.

The term "reduced tumorigenicity" was introduced to describe cells that have been transformed in less tumorigenic or neuturogena cells. Cells with reduced tumorigenicity or do not form tumors when introduced in vivo, or lagered before the onset of tumor growth in vivo is weeks or months and/or growth of tumor mass, defined in three dimensions, is slower compared with tumors in which the gene is a tumor suppressor inactivated or failed.

The term "effective amount" means that amount of a vector or anti-cancer protein, which allows to achieve a positive result in the control of cell proliferation. For example, one dose , one dose daily for five days. The effective amount can vary depending on the nature of the disease, the patient's condition and other factors well known in the prior art. The effective amount can be easily determined by an experienced researcher.

In the scope of the present invention also includes a method of facilitating disease, characterized by the presence of a hyperproliferative cells or genetic defect, by introducing to the subject an effective amount of the vector described above and containing the foreign gene product which is able to reduce the severity of the disease under appropriate conditions. The term "genetic defect" means any disease or abnormality caused by inherited factors, such as sickle cell anemia or disease Tay-Sachs.

The present invention relates also to a method of reducing proliferation of tumor cells in a subject by introducing into the tumor mass effective amount of adenoviral expression vector containing antitumor gene that is different from suppressor gene tumor. Antitumor gene may encode, for example, timedancing (TC). Then the subject is administered an effective amount of a therapeutic agent,s such therapeutic agents are metabolites timedancing, for example, ganciclovir (GCV), 6-methoxypurine arabinopyranoside (araM) or their functional equivalents. For the manifestation of toxic effects to the cell master gene timedancing and metabolite timedancing must use competitive. However, in the cell GCV fosfauriliruetsa and becomes a potent inhibitor of DNA synthesis, and araM turns into toxic anabolic araATP. To reduce the proliferation of tumor cells can be used, and other antitumor genes in combination with the appropriate therapeutic agents. Such a combination of genes and therapeutic agents are well known in the prior art. Another example can be claimed in the present invention, the vector expressing the enzyme sitoindosides. Such a vector may be used in combination with the drug 5-fluorouracil (Austin and Huber, 1993). Another example is the combination of a recently discovered gene Deo-Delta E. coli 6-methyl-purine-2'-deoxynucleosides (Sorscher et al., 1994).

Using the above-described genes tumor suppressor, as well as the use of other anti-cancer genes both by themselves and in combination with an appropriate therapeutic agent, allows you to influence nekontroliruem the persons therapy to stop uncontrolled cell growth in a patient, which leads to relief of symptoms and alleviate the patient's cachexia. The results of such therapy are expressed (but are not limited to specified) in the increased life of the patient, reducing tumor mass, the apoptosis of tumor cells or reducing the number of circulating tumor cells. Methods of assessment positive effects of this therapeutic approach is well known from the prior art.

The object of the present invention is a recombinant adenoviral expression vector, characterized by partial or complete deletion of the DNA adenovirus protein IX, and carries a foreign gene that encodes a foreign protein, despite the fact that alien protein is the product of a suicide gene or its functional agent. The above antitumor gene TC is an example of a suicide gene, because the product of its expression is or can be lethal to cells. For TC lethality induced by the presence of GCV. Gene TC derived from herpes simplex virus using methods well known in the prior art. For the purposes of the present invention the source of the gene timedancing (TC) virus, a simple repneca (HSV-1) is plasmide/BKTK in cells of E. coli HB 101 (ATCC # 39369). Danovaroussinova of the expression vector in combination with appropriate pharmaceutically acceptable carrier. The introduction can be carried out, for example, by direct injection into the tumor mass. When such specific neoplasms as hepatocellular carcinoma (HCC), you can use the introduction into the hepatic artery as the blood supply of the majority of HCC is performed it is through this artery. To control the proliferation of tumor cells and reduce tumor mass is induced cell death by introducing patients metabolite TC, such as ganciclovir (GCV). Metabolite LC can be introduced systemically, locally in the tumor or in the case of HCC, injected into the hepatic artery. Metabolite TC preferably daily, but depending on the circumstances, the frequency of injections can be increased or reduced. Metabolite TC may be injected simultaneously or sequentially with the introduction of TC - containing vector. Experienced researchers can determine the dose and duration of administration, which are therapeutically effective.

How tumor-specific delivery suppressor gene tumor can be carried out while ensuring contact the target tissue of this animal with an effective amount of a recombinant adenoviral vector expression, as claimed in the present invention. GBV. "Collateral contact" means any method of effective delivery vector, for example, vnutriplevralnoe introduction.

The use claimed in the present invention adenoviral vector for the preparation of medicines intended for the treatment of certain diseases, is also the subject of the present invention.

Below are examples to illustrate but not to limit, the scope of the present invention.

EXPERIMENT N I

For these studies as a starting material was selected plasmid pAd/MLP/p53/E1b-. This plasmid is based on pML2, derived from pBR322 (pBR322 with a deletion of bases 1140-2490), and contains sequences of adenovirus type 5 with 1 to 5788-th pair of bases with a deletion of bases 4357-3327. Instead deletion of Ad5 357-3327 entered transcription cassette consisting of the late promoter of Ad2, trinomial leader cDNA Ad2 and p53 cDNA person. Thus, the typical vector with the replacement of the E1 sequences, deletion of genes E1a and E1b Ad5, but containing the gene of the protein IX Ad5 (see review of adenoviral vectors: Graham and Prevec (1992)). The Ad2 DNA was obtained from Gibco BRL. The restriction enzyme and T4 DNA ligase were obtained from New England Biolabs. Comp is Tur (ATCC). Resin for purification of DNA Prep-A-Gene was purchased from a company BioRad. LB-broth for cultivation of bacteria was purchased from Difco company. Column Quagen for DNA extraction were purchased from firms Quagen, Inc. Virus Ad5 dl327 was obtained from R. J. Schneider, NYU. Set for transfection of DNA MBS was bought by the company Stratagene.

One (1) microgram pAd/MLP/p53/E1b - restrictively 20 units of each enzyme Ecl 136II and NgoMI in accordance with the manufacturer's recommendations. Five (5) µg Ad2 DNA was restrictively 20 units of each of the enzymes DraI and NgoMI in accordance with the manufacturer's recommendations. Restriction of the mixture were applied in separate wells of a 0.8% agarose gel and subjected to electrophoresis at 100 volts for 2 hours. Restriction fragment pAd/MLP/p53/E1b - 4268 KB and restriction fragment 6437 KB Ad2 were isolated from the gel using a resin for the extraction of DNA Prep-A-Gene in accordance with the manufacturer's recommendations. Restriction fragments were mixed and treated with T4 DNA ligase in a total volume of 50 µl with the 16oC for 16 hours according to the manufacturer's recommendations. After legirovaniem 5 ál of reaction mixture was used for transformation of E. coli cells OH5-alpha (selection were resistance to ampicillin). Six bacterial colonies obtained as a result of this the 37oC with constant shaking. According to standard methods (Sambrook et al., 1989) of each bacterial culture was isolated plasmid DNA. The fourth part of each sample of plasmid DNA was subjected to restriction 20 units endonuclease Xhol for selecting the "right" recombinant containing restriction fragments Xhol in 3627, 3167 and 1445 base pairs. Five of the six specimens tested contained the desired recombinant. One of these clones was used for inoculation of 1-liter culture in LB medium and the subsequent release of large quantities of plasmid DNA. After incubation over night from 1-liter culture was isolated DNA using Qiagen columns according to the manufacturer's recommendations. The obtained plasmid was designated Pad/MLP/p53/PIX. The designs of these plasmids have been deposited in the American Collection of Cell Cultures (ATCC), 12301 Parklawn Drive, Rockville, Maryland, USA, 12301, October 22, 1993. The Deposit made under the terms of the Budapest agreement concerning the international Deposit of microorganisms for the purposes of patenting. This Deposit is received a number ATCC 75576.

To construct a recombinant adenovirus, 10 μg of the plasmid Pad/MLP/p53/PIX-linearizable 40 units restrictase EcoRI. DNA of adenovirus 5-th Puyraveau in the sucrose gradient. Ten (10) μg restricciones EcoRI plasmid Pad/MLP/p53/PIX - and 2.5 µg restricciones Clal Ad5 DNA dl327 were mixed and used for transfection of approximately 106the 293 cells using a set of MBS for transfection of mammalian cells according to the manufacturer's recommendations. Eight (8) days after transfection of 293 cells subcultured at a ratio of 1: 3 in fresh medium, and after 2 days on transfected cells became visible cytopathic effect induced by adenovirus. On the 13th day after the transfection of cells by a standard method (Graham and Prevec, 1991) was isolated DNA and restriction analysis was performed using restrictase Xhol. The expression of p53, caused by a virus, verify the subsequent infection of cells of osteosarcoma Saos-2 virus-containing lysate and Western blot turns with anti-p53 monoclonal antibody 1801 (Novocasta Lab.Ltd., UK).

EXPERIMENT N II

Materials and methods

Cell line

Recombinant adenoviruses were grown and accumulated in the cells 293 (embryonic kidney human ATCC CRL 1573 grown on medium DME with 10% calf serum (Hyclone). Cells Saos-2 were grown on medium käina with the addition of 15% fetal calf serum. Cells HeLa and Hep 3B were grown on medium with DME dobavlennuju calf serum. Cells Saos-2 were kindly provided by Dr. Eric Stanbridge. All other cell lines were obtained from ATCC.

Construction of recombinant adenoviruses

To construct Ad5/p53 viruses 1.4 kielbasy Hindlll-Smal fragment containing the full-size cDNA of p53 (table 1), was isolated from the plasmid pGEM1-p53-B-T (courtesy of Dr.Wen Hwa Lee) and inserted into the cloning site of the expression vector pSP72 (Promega) using standard cloning techniques (Sambrook et al., 1989). Insert p53 was isolated from this vector after restriction Xhol-Bglll and subsequent gel electrophoresis. Then coding sequence of p53 was built in adenoviral vectors for gene transfer pNL3C or pNL3CMV (courtesy of Dr.Robert Schneider), which contain inverted terminal repeat Ad5 5' viral signals packing, E1a enhancer and the main promoter of the late proteins of Ad2 (MPL) or the promoter of the early gene of human cytomegalovirus (CMV), followed by a three-membered leader cDNA and sequence 3325-5525 Ad5 in the main part PML2. These new constructs region Ad5 E1 (360-3325 base pairs) replaced by p53 under the control of the Ad2 MPL (A/M/53) or CMV person (A/C/53), and in both cases for p53 should trinomial leader DVS is leitlinie recombinants with p53 under the control of the MPL or CMV (A/M/N/53, A/C/N/53), which had a deletion 705 nucleotide sequences of Ad5 to remove the coding region of the protein IX (PIX). In the quality control of the original plasmid pNL3C was obtained recombinant adenovirus without inserting p53 (A/M). The other served as control recombinant adenovirus, a gene encoding beta-galactosidase under the control of CMV (A/C/beta-Gal). Plasmids were linearizable restriction Nrul or EcoRI and cotranslationally with a large fragment of Clal-restricciones mutant Ad5 or dl309 dl327 using the kit for the Ca/PO4-transfection (Stratagene). Allocated viral plaques and recombinants were identified by restriction analysis and PCR using recombinant specific primers to sequences trinomial leader DNA located "below" cDNA sequences of p53. Then the recombinant virus was purified by the method of finite dilutions, were isolated viral particles and was titrated by standard methods (Graham and van der Eb, 1973; Graham and Prevec, 1991).

Detection of p53 protein

Cells Saos-2 or Hep3B (5105) infected the indicated recombinant adenoviruses for 24 hours with increasing multiplicity of infection (plaque-forming units of virus per cell). Then the cells once opola the ml Aprotinin, 10 µg/ml leupeptin and 1 mm PMSF. Cellular proteins (approximately 30 µg) were separated in 10% SDS-PAGE and transferred to nitrocellulose. Membranes were incubated with anti-alpha-p53 antibody PAb 1801 (Novocastro), and then conjugated sheep artemisinin lgG to horseradish peroxidase. P53 protein was visualized by chemiluminescence (set ECL, Amersham) with a film Kodak XAR-5.

Determination of the rate of DNA synthesis

Cells (5103/well) were seeded in cell 96-well plates (Costar) and allowed to attach overnight (37oC, 7% CO2). Then cells within 24 hours of infected particles purified recombinant virus multiplicity of infection of 0.3 to 100. 24 hours after infection were replaced medium and the incubation continued for up to 72 hours. For 18 hours before harvesting the cells was added 3H-thymidine (Amersham, 1 µci/well). Cells were besieged on fiberglass filters and determined the level of bound peroxidase radioactivity using a scintillation beta-counter. The incorporation of 3H-thymidine was expressed as a percentage (+/- the average deviation from control environment and set in dependence on the multiplicity of infection.

Tumorogenesis for naked mice

Approximately 2.4108cells Saos-2 growing on bottles T, was treated with BU is Bali with the virus during the night cells were injected subcutaneously into the left and right side atipicheskim naked BALB/c mice (4 mice per group). One side was injected cells treated with virus A/M/N/53, and the other with a virus A/M, thus, each mouse served to control for experience. Animals received bilateral injections of cells treated with buffer, which served as an additional control. The tumor size (length, width, and height) and body weight were determined twice a week for 8 weeks. The volume of tumors was determined for each animal based on the spherical geometry of the tumor, despite the fact that the radius took one second from the average of all three dimensions of the tumor.

Analysis of tumor RNA

Atipicheskim naked BALB/c mice (approximately 5 weeks of age) were injected subcutaneously into the right side 1107cells H69 (small cell carcinoma of the lung). Tumors were allowed to grow for 32 days, by this time they had reached a volume of 25-50 mm3. Mice were performed peritumoral injection of recombinant adenoviruses A/C/53 or A/C/beta-Gal (2109plaque-forming units, PFU) into the subcutaneous space below the tumor mass. The tumor was cut out on the 2nd and 7th days after injection of adenoviruses and rinsed in PBS. Tumor homogenized and isolated them from the total RNA using a set of TriReagent (Molecular Research Center, Inc.,). was olovely to determine the expression of p53 mRNA using RT-PCR (Wang et al., 1989). Primers were designed to americasouth sequence between adenovirus trinomial leader cDNA and located "below" p53 cDNA. Thus was achieved by amplification of only recombinant, but not endogenous p53 sequences.

Hemotherapy tumors in Nude mice by p53

Approximately 1107tumor cells H69 (SCLC) in a volume of 200 µl was injected subcutaneously to female animicheskih naked BALB/c mice. Tumors were formed within 2 weeks and then animals are randomized by the size of the tumors (N= 5/group). Conducted peritumoral injection of adenovirus A/M/N/53 or control adenovirus A/M (2109PFU/injection) or buffer (1% sucrose in PBS) twice a week, a total of 8 doses per group. Within 7 weeks, twice a week was determined by the tumor size and body weight; the volume of the tumors was assessed as described previously. In addition, we determined the effect of therapy on survival of mice.

Results

Construction of recombinant RS-adenovirus

Containing the p53 adenovirus was constructed by replacing part of the region E1a and E1b of adenovirus type 5 on p53 cDNA under the control of the promoter of Ad2 MPL (A/M/53) or CMV (A/C/53) (schematically represented at f the m multiply only in 293 cells, which serve as a source of gene products El Ad5 "TRANS" type (Graham et al., 1977). After identification of recombinant adenoviruses as restriction analysis and PCR, the full cDNA of p53 one of the recombinant adenoviruses (A/M/53) was prosecution in order to verify the absence of mutations. Then purified preparations p53-recombinants were used for infection of HeLa cells with the aim of detection of wild-type adenovirus. HeLa cells, which are supermassive for replication of E1-deletirovanie adenovirus, were infected 1-4109infectious units of recombinant adenovirus, then they were cultured for 3 weeks and observed the appearance of cytopathic effect (JRC). This replication of recombinant adenovirus or contamination by wild-type virus was not detected. In cultures of control cells infected with wild-type adenovirus, JRS was obvious, and the sensitivity of this method is approximately 1 out of 109.

Expression of p53 protein using recombinant adenovirus

In order to determine, whether Express recombinant p53-adenovirus p53, these viruses were infected cell line, which does not expressely carcinoma) were infected for 24 hours p53 recombinant adenovirus A/M/53 or A/C/53 with a multiplicity of infection ranging from 0.1 to 200 PFU/cell. Western analysis of lysates prepared from infected cells showed dose-dependent expression of p53 in both cell types (Fig. 3). In uninfected cells, p53 has not been detected. The levels of endogenous p53 wild-type normal very low and almost not detected by Western analysis of cell lysates (Bartek et al., 1991). However, it is clear that p53 wild-type easily detected after infection with the virus A/M/53 or A/C/53 with a low multiplicity of infection (Fig. 5), suggesting that even small doses of p53 recombinant adenovirus capable of producing potentially effective amount of p53.

p53-dependent changes in morphology

Reintroduction of p53 wild-type p53-negative cell osteosarcoma line Saos-2 resulted in a characteristic increase and flattening these are usually spindle-shaped cells (Chen et al., 1990). Subconfluent cells Saos-2 (1105/ 10 cm Cup) were infected with a multiplicity of infection of 50 virus A/C/53 or a control virus (A/M and incubated at 37oC for 72 hours. By this time, the control uninfected cells formed a monolayer. At this time the expected morphological changes became clearly visible in the cups with cells treated A/C/53 (Fig. 6, pig. 6, panel B). This effect is not a function of the density of cells as in the control Cup cells, seeded with lower density, maintained their normal morphology to 72 hours of incubation, when their density was similar to that for A/C/53-infected cells. Earlier results indicate high levels of expression of p53 protein in cells Saos-2 at a multiplicity of infection of 50 (Fig. 5A), and also confirm the fact that p53 protein expressed using recombinant adenoviruses, is biologically active.

Inhibition of the synthesis of cellular DNA by p53

For further study RS recombinant adenoviruses were tested by incorporating labeled 3H-thymidine their ability to inhibit the proliferation of human tumor cells. Previously it was shown that the introduction of the p53 wild-type cells that do not Express endogenous p53 wild type, can stop cells at the transition from the G1 phase to the S phase of the cell cycle, which leads to the suppression of incorporation of labeled thymidine into newly synthesized DNA (Baker et al. , 1990; Mercer et al., 1990; Diller et al., 1990). Various tumor cell lines deficient in p53, adenovirus infected vectors A/M/N/53, A/C/N/53 or not expressing the infection with recombinant viruses A/M/N/53 and A/C/N/53 in the case of seven of the nine tested tumor cell lines (Fig. 7). Both construct were able to inhibit DNA synthesis in these tumor cells, regardless of whether expressed they mutant p53 or not expressed p53. In this experiment it was also shown that the construct of A/C/N/53 is significantly more efficient than A/M/N/53. In cells Saos-2 (osteosarcoma) and MDA-MB468 (breast cancer) when infection construct A/C/N/53 with a plurality of not higher than 10 was achieved almost 100% inhibition of DNA synthesis. At those doses, when the suppression of DNA synthesis control adenovirus was reached only 10-30% was observed 50-100% inhibition of DNA synthesis when using any of p53 recombinant adenovirus. On the contrary, any one construct was not observed any p53-specific effect in comparison with the control virus in the case of cells Hep G2 (cell line hepatocarcinoma expressing endogenous p53 wild-type, Bressac et al., 1990) and leukemic cell line K562 (p53-null).

Tumorogenesis for naked mice

B more stringent test on the functioning of recombinant adenoviruses containing p53, tumor cells were infected ex vivo and then injected naked mice to test the ability of recombinant suppress tumor growth in vivo.Dili in different flanks naked mice. Then for 8 weeks twice a week to determine the size of tumors. When the multiplicity of infection equal to 30, none of the animals the growth of p53-treated tumors was not observed, despite the fact that the growth of control tumors occurred normally (Fig. 8). The progressive increase of the tumors treated with the control virus was similar to that observed in animals treated only with buffer. Observed clear differences in the growth of tumors in the case of control adenovirus and p53 recombinant adenovirus at a multiplicity of infection of 3, although 2 of the 4 p53-treated mice, tumor growth began about 6 weeks. Thus, recombinant adenovirus A/M/N/53 conducive to a p53-specific suppression of tumors in vivo.

Expression of Ad/p53 in vivo

Despite the fact that treatment of tumor cells ex vivo followed by the introduction of their animals is an important test for tumor suppression, more important in clinical terms are presented experiments on the introduction of p53 recombinant adenovirus and the expression of p53 in tumors that developed in vivo. With this purpose, cells H69 (SCLC, p53 - null) were injected subcutaneously naked mice and allowed to form tumors within 32 days. At this time, once the after introduction of the virus in the tumor were cut from each tumor were allocated the poly RNA. For detection of mRNA of p53 in p53-treated tumors used RT - PCR with primers specific regarding recombinant p53. Signal p53 was not detected in tumors, cut in animals treated with virus with beta-galactosidase (Fig. 9, lines 3 and 6). Amplification with actin primers served as a control in the reaction RT-PCR (Fig. 9, lines 7-9), and a plasmid containing the sequence of recombinant p53, served as a positive control for p53-specific bands (Fig. 9, line 10). This experiment shows that p53 recombinant adenovirus can specifically direct the expression of p53 mRNA in developed tumors after a single injection in the peritumoral space. It also demonstrates the persistence of the virus in vivo for at least a week after infection p53 recombinant adenovirus.

The efficiency of in vivo

To determine the validity of gene therapy developed tumor model was used naked mice - carriers of the tumor. In the right side naked mice were injected subcutaneously H69 cells and allowed the tumors to grow for 2 weeks. Then the mice twice a week peritumoral were injected with buffer or recombinant virus (8 injections). On protage as tumors in mice the treated virus A/M/N/53, grew much more slowly (Fig. 10A). After termination of the injection control tumors continued to grow, and tumors treated p53, grew marginally or not grew quite at least a week in the absence of additional exogenous p53 (Fig. 10A). Despite the fact that the control animals received only buffer was observed accelerated growth of tumors in comparison with any group of animals treated with virus, a significant difference in body weight between the three groups for all the processing time was found. Pitting tumors in some animals reduced the validity of the definitions of the tumors after the 42nd day. However, continued monitoring of animals to determine the survival time showed an advantage in the survival of p53-treated animals (Fig. 10B). The last of the processed control adenovirus animal died on the 83rd day, whereas all control animals received only buffer, died to the 56 th day. On the contrary, all five animals treated with virus A/M/N/53, survived (130-th day after injection of cells) (Fig. 10B). The combination of these data confirms the specific influence of p53 on tumor growth and survival time in animals with developed p53-definitie adenoviral vectors, capable of high expression levels of p53 wild-type dose-dependent manner. Each vector contained deletions of regions of E1a and E1b, which made the virus replication defective (Challberg and Kelly, 1979; Horowotz, 1991). It is important to note that these deletions were captured sequences encoding proteins of 19 KD and 5 KD E1b. It is shown that the 19 KD protein involved in inhibition of apoptosis (White et al., 1992: Rao et al., 1992), and the protein of 55 KD able to contact the protein p53 wild-type (Samow et al., 1982; Heuvel et al., 1990). Deleted data adenoviral sequences are removed potential inhibitors of p53 function, able to act by direct binding of p53 or by inhibiting p53-mediated apoptosis. Received additional constructs that were deleterows remaining 3' E1b sequences, including the sequence encoding the protein IX. Although, as shown, deletions in region E3 lead to loss of capacity of the vector is approximately 3 KB in comparison with wild-type adenovirus (Ghosh-Choudhury et al. , 1987), similar deletions were made in the constructions A/M/N/53 and A/C/N/53, so that the capacity of these vectors is within the specified limits. Deleterevision region pIX content adenoviral sequences, pomologiczna by recombination competent for replication of wild-type adenovirus. Constructs, lost the coding sequence pIX had the same efficiency as compared to constructs with the given sequence.

The effectiveness of p53 adenovirus in vitro

In accordance with the strict dozozawisimuu the expression of p53 in infected cells revealed a dose-dependent p53 - specific growth suppression of tumor cells. In tumor cells of various types, which are not expressively p53 wild-type, was suppressed cell division, as shown by a decrease in DNA synthesis. Recently Bacchetti and Graham (1993) in similar experiments showed p53-specific suppression of DNA synthesis in cell lines carcinoma ovarian SKOV-3 by recombinant p53 adenovirus. The inhibition of growth using claimed in the present invention p53-recombinants has been shown not only to carcinoma of the ovary, but also in several other tumor cell lines of the person presenting clinically important human neoplasias, including lines, in which there is increased expression of mutant p53 protein. When the multiplicity of infection, when the recombinant A/C/N/53 showed 90-100% efficiency of suppression of DNA synthesis in the tumor data types, activity controling is no cause differentiation and increase in the proportion of cells in G1 with respect to the S+G2 for leukemia cells K562, the authors of the invention p53-specific effect on these cells was not observed. Horvath and Weber (1988) reported that lymphocytes of peripheral blood in a high degree of nopermission for adenoviral infection. In some experiments, recombinant adenovirus A/C/beta-Gal hardly infected unresponsive K562 cells, while other cell lines, including control cell line Hep G2 cells with a pronounced effect of p53 that are easily infected. Thus, at least part of the variation in efficiency is due to differences in the infectivity of the virus, although other factors may also be relevant.

The results obtained with virus A/M/N/53 and shown in Fig. 8, show that in vivo may complete suppression of tumor growth. The resumption of growth of tumors in two animals of the four-treated p53 with a low multiplicity of infection, due to the low percentage of cells infected with the p53 recombinant at this dose. Complete suppression of tumor growth in the case of the influenza A/M/N/53 at the high dose, however, shows that the ability to resumption of tumor growth can be overcome.

The effectiveness of p53 adenovirus in vivo

Given saweka, in which there is no expression of p53 wild-type, can be treated ex vivo p53, which leads to suppression of tumor growth in subsequent introduction of animal cells. The authors have provided the first proof of the possibility of gene therapy of tumors by moving suppressor gene in tumors that developed in vivo. This method leads to suppression (suppression) of tumor growth and increase survival time. In the system described by the authors, shipping agent in tumor cells is not based on direct injection into the tumor mass. In contrast, p53 recombinant adenovirus was injected into the peritumoral space and a tumor was found in the expression of p53 mRNA. Expressed by recombinant p53 was functional and significantly inhibited tumor growth compared with control tumors treated with adenoviruses, not expressing p53. However, suppression of tumor growth was observed in both groups as treated p53-containing and p53-nesteriak viruses, in contrast to the control (treatment only buffer). It was shown that the naked mice local expression of tumor necrosis factor (TNF), interferon-gamma, interleukin-2 (IL-2), interleukin 4 or 7 (IL-4, IL-7) may cause temporary with the first inductor of interferon alpha and beta (see review Gooding and Wold, 1990). Thus, it is not surprising that some suppression of tumors in Nude mice was observed in case of control adenovirus. This virus-induced suppression of tumors was not observed in the case of tumor cells Saos-2, treated ex vivo with control virus. P53-specific inhibition of tumors in vivo is particularly evident when the long-term monitoring of animals (Fig. 10). The time of survival of p53-treated mice was dramatically increased, so 5 of the five experimental animals were alive after 130 days after injection of cells compared to 0 out of five treated with control adenovirus. In surviving animals tumor growth continued, which, apparently, is due to the fact that not all cells were initially infected with p53 recombinant adenovirus. This circumstance can be overcome by higher doses of virus, or more frequent injections. In addition, off promoter (Palmer et al.,1991) or additional mutations can cause resistance of these cells to process the p53 recombinant adenovirus. For example, mutations in a recently described gene WAF1, which is induced by p53 wild-type and then suppresses the transition of cells in S-phase of the cell cycle (El-Deiry et al., 1993; H is the iMER shows the use of suicide genes and tissue-specific expression of these genes in gene therapy approaches, described in this invention. As the target was selected hepatocellular carcinoma, as it is one of the most common neoplasms of the person and leads to the deaths of about 1250000 of people around the world every year. The frequency of this cancer is particularly high in South-East Asia and Africa, where it is associated with infection with hepatitis B and C and exposure to aflatoxin. The only treatment for hepatocellular carcinoma on today is surgical, although less than 20% of patients are considered candidates for surgery (Ravoet S. et al. , 1993). However, the ways to reduce the proliferative capacity of tumor applicable to other neoplasms other than hepatocellular carcinoma.

Cell line

All cell lines except line HFL were obtained from American Cell culture Collection (ATSS), 12301 Parklawn Drive, Rockville, Maryland. The code numbers of ADS given in parentheses. Cell line embryonic human liver 293 (CRL-1573) was used to obtain and capacity of recombinant adenoviruses. Cells were grown on medium D with the addition of 10% calf serum (Hyclone). Cell lines hepatocellular carcinoma Hep 3B (HB-8064), Hep G2, HB 8065) and HFL supported (HTB 132) and BT-549 (HTB 122). Hepatic cell line Chang (CCL 13) were grown in MEM medium with addition of 10% fetal calf serum. Cell line HLF was obtained from Dr. ditch T. Morsaki and H. Kitsuki (medical faculty of the University of Kyushu, Japan).

Construction of recombinant virus

Two adenoviral expression vector, designated here as ACNTK and AANTK deprived of the function of the protein IX (shown in Fig. 11) capable of expression of the suicide gene timedancing (TC) in tumor cells. Third adenoviral expression vector, designated AANCAT, was designed to further demonstrate the feasibility of specific targeted gene expression in specific types of cells using adenoviral vectors. Data adenoviral constructs "collected", as shown in Fig. 11 and 12, and they are derived from those structures that were previously described for the expression of genes tumor suppressor.

For expression of the transferred gene was used expression cassette, in which the transcription of the TK gene or gene chloramphenicolchloramphenicol (CAT) was used early promoter/enhancer of the human cytomegalovirus (Boshart, M. et al., 1985) or enhancer/promoter, alpha-fetoprotein cloudlinux types, and the constructs with the promoter/enhancer AFP is expressed specifically in cells of hepatocellular carcinoma (HCC), in which the expression of AFP was observed in 70-80% of patients with this type of tumors. In construct with a promoter/CMV enhancer to enhance broadcast transcript TC used trinomial leader sequence of adenovirus type 2 (Berkner, K. L and Sharp, 1985). In addition to deletions of E1 in both adenoviral vectors deleterule 1.9 KB DNA region E3. DNA deleteriously in the E3 region is not essential for virus replication and such deletion increases by an equivalent amount (1.9 KB) the capacity of the recombinant virus in relation to alien DNA (Graham and Prevec, 1991).

To demonstrate the specificity of the promoter/enhancer AFP was designed virus AANCAT in which the marker gene chloramphenicolchloramphenicol (CAT) placed under the control of promoter/enhancer AFP. In viral construct ACNTK trinomial leader sequence Ad2 placed between genome TC and promoter/CMV enhancer. It is shown that the three-membered leader enhances the transmission connecting genes. Replacement in the field E1 interfere with the ability of recombinant viruses for replication, limiting their reproduction only ctor ACNTK: plasmid pMLBKTK in E. coli HB 101 (from ATS, # 39369) was used as the source of the gene timedancing (TK) of herpes simplex virus (HSV-1). Gene TC was cut from the plasmid in the form of a fragment of 1.7 KB with restriction enzymes Bgl II and Pvu II, and then subcloned sites BamHI and EcoRV in plasmid pSP72 (Promega) using standard cloning techniques (Sambrook et al., 1989). The insertion of the TK-gene was then excised from this vector in the form of a 1.7 KB fragment of restrictase Xba 1 and Bgl II and cloned into a plasmid pACN (Wills et al., 1994). Twenty (20) micrograms of this plasmid, designated pACNTK, linearizable using restrictase EcoRI and cotranslationally in 293 cells (ATCC CRL 1573) with 5 μg of digested Cla I plasmids ACBGL (Wills et al., 1994 supra) and a set for the calcium-phosphate transfection (Stratagene, San Diego, California). Isolated viral plaques and identified recombinants ACNTK using restriction analysis of isolated DNA. When used restrictase Xhol and BsiWI. Positive recombinants were then purified by the method of finite dilution, increased and was titrated by standard methods (Graham and Prevec, 1991).

Adenoviral vector AANTK: the promoter of the gene alpha-fetoprotein (AFP-P) and enhancer gene (AFP-E) were cloned from human genomic DNA (Clonetech) by PCR amplification using p is s) contained a Nhel restriction site in the 5'-primer and Xbal linker, Xhol, Kpnl 3'-primer. The sequence 5'- primer was as follows: 5'-CGC GCT AGC TCT GCC CCA AAG AGC T-C'. The sequence 3'-primer was as follows: 5'-CGC GGT ACC CTC GAG TCT AGA TAT TGC CAG TGG TGG AAG-3'. The primers used for marquee AFE (1763 base pairs), had a Notl restriction site at the 5'-primer and a Xbal restriction site at the 3'-primer. The sequence 5'-primer was as follows: 5'-CGT GCG GCC GCT GGA GGA CTT TGA GGA TGT CTG TC-3'. The sequence 3'-primer was as follows: 5'-CGC TCT AGA GAG ACC AGT TAG GAA GTT TTC GCA-3'. For PCR amplification DNA was denaturiruet 7 minutes at 97oC, followed by 5 cycles of amplification at 97oC 1 minute, 53oC 1 minute, 72oC for 2 minutes, and finally, extension at 72oC for 10 minutes. Amplificatory fragment AFE was restrictively enzymes Notl and Xbal and was built on the site Notl and Xbal in plasmid vector pA/1TR/B, containing sequences of adenovirus type 5 (1-350 and 3330-5790), separated by polylinker containing a Notl restriction enzymes cut sites, Xhol, Xbal, Hindlll, Kpnl, BamHI, Ncol, Smal and Bgl II. Amplificatory fragment AFP-E digested by enzymes Nhel and Kpnl, and was built in containing AFP-E construct described above by restriction enzymes cut sites of these enzymes. This new construct was then restrictively Fe is a COP Xbal-NgoMl plasmids pACN, containing nucleotides 4021-10457 adenovirus type 2, to obtain plasmid pANN, containing both the promoter and enhancer of alpha-fetoprotein. This construct was then restrictively enzymes EcoRI and Xbal to highlight the fragment size 2.3 KB containing the inverted terminal repeat of Ad5 virus, AFP-E and AFP-P, which is then ligated with EcoRI-Xbal 8.5 - KB fragment described above plasmids pACNTK obtaining plasmids pAANTK, in which the TK gene in adenovirus environment placed under the control of the promoter and enhancer of the gene alpha - fetoprotein. This plasmid was then linearizable using EcoRI and cotranslational together with the large fragment of Clal restricciones plasmids ALBGL, as described above, and the recombinants identified AANTK, were isolated and purified as described above.

Adenoviral vector AANCAT: gene chloramphenicolchloramphenicol (CAT) was allocated from the Basic Vector pCAT (Promega Corporation) with the last restriction enzymes Xbal and BamHI. Received 1.64 - KB fragment ligated with restricciones Xbal and BamHI the plasmid pAAN (described previously) to obtain plasmid pAANCAT. This plasmid was then linearizable using EcoRI and cotranslationally with a large fragment restricciones Clal rA/C/beta-gal with poluchenii AANCAT.

The reporter gene expression: akinsete (Costar) and left overnight to attach (37oC, 7% CO2). Virus infection ACBGL conducted during the night when the multiplicity of infection equal to 30. After 24 hours the cells were fixed with 3.7% formaldehyde, washed PBS and stained with the reagent X-gal (1 mg/ml, USB). The results were expressed in the adopted symbols(+, ++, +++), determining the percentage of stained cells for each multiplicity of infection. [+=1-33%, ++=33-67%, and +++67%).

The reporter gene expression: expression of the CAT

Cells HepG2, Hep B3, HLF, Chang and MDA-MB468 seeded by 2106on 10-cm cups (three cups each culture) and incubated overnight at 37oC, 7% CO2. Then the cells on each plate were infected or AANCAT with a multiplicity of infection of 30 or 100, or left uninfected, and incubated for 3 days. Then the cells were removed with trypsin, washed PBS and suspended in 100 μl of 0.25 M Tris-HCl pH 7.8. The three samples were frozen and thawed, the supernatant was transferred into a new tube and incubated at 60oC for 10 minutes. Then the samples were centrifuged at 4oC 5 minutes and supernatant determined the protein concentration by Bradford (Bio-Rad Protein Assay Kit). The protein concentration in the samples was aligned, bringing the final volume up to 75 ál using 0.25 M Tris, 25 μl of 4 mm acetyl-COA and 1 mm14C-chloramphenicol. Samples were inkubirovaniya 5 minutes at room temperature. The upper phase was transferred into a new tube and evaporated ethyl acetate by centrifugation under vacuum. The reaction products were dissolved in 25 μl of ethyl acetate and put it on a plate for thin-layer chromatography (TLC). The plate was placed in a balanced (95% chloroform, 5% methanol) chamber for TLC. The solvent was allowed to migrate in the upper part of the chamber, and then the plate was dried and exposed to x-ray film.

Cell proliferation: enable3H-thymidine

Cells were seeded at 5103cells/cell in the cell 96-well plates (Costar) and incubated overnight (37oC, 7% CO2). Serially diluted viruses ACN, ACNTK or AATK environment DM with 15% fetal calf serum and 1% glutamine was used to infect cells with a multiplicity of infection equal to 30. Incubation with the virus continued overnight, after which the cells were added ganciclovir in logarithmic dilutions ranging from 0.001 to 100 μm (micromoles). For 12-18 hours before harvesting the cells in each well was added 1 µci 3H-thymidine (Amersham). After 72 hours after infection the cells were besieged on fiberglass filters and determined the number of enabled1H-thymidine method liquid scintillation (TopCount, Packard). the second dose (ED50 +/- standard deviation) for 50% reduction of cell proliferation over the control environment. ED50 values were estimated by matching with the logical equation "dose-response".

Cytoxicity: release of lactate dehydrogenase (LDH)

Cells (HFL, human hepatocellular carcinoma, HCC) after infection ACN or ACTK was treated with ganciclovir, as described in the section "cell Proliferation". After 72 hours after addition of ganciclovir cells were centrifuged and supernatant was removed. Levels of lactate dehydrogenase was determined colorimetrically (Promega, Cytotox 96TM). The mean LDH release (+/- mean deviation) were placed in dependence on the multiplicity of infection.

Therapy in vivo

Cells hepatocellular carcinoma human (Hep 3B) was administered subcutaneously ten females animicheskih mice nu/nu (Simonsen Laboratories, Gilroy, CA). Each animal received in the left side about 1107cells. Tumors were allowed to grow for 27 days, and then mice randomized according to tumor size. The mice were injected every other day intratumoral or peritumoral virus ACNTH or control virus ACN (1109infectious units per 100 μl). Just each mouse received three injections. 24 hours after the first injection of adenovirus to mice administered intraperitoneally injected ganciclovir (Cytovene, 100 mg/kg). These injections were performed daily the three measurements using calibrated calipers and the volume of the tumors was determined by the formula: 4/33where r is one half the average size of the tumor.

Results

Recombinant adenoviruses were used to infect three cell lines hepatocellular carcinoma (HLF, Hep3B and Hep-G2). As controls were used cell line human liver (Chang) and two lines of breast cancer (MDAMB468 and BT549). To demonstrate the specificity of the promoter/enhancer AFP was designed virus AANCAT. This virus was used to infect cells that Express (Hep 3B, Hep-G2) or does not Express (HLE, Chang, MDAMB468) marker of hepatocellular tumors alpha-fetoprotein (AFP). As shown in Fig. 13, AANCAT directs the expression of the marker gene CAT only in HCC cells, which are capable of expression of AFP (Fig. 13). Efficiency ACNTH and AANTK for the treatment of HCC was tested using the inclusion of 3H-thymidine to determine the effect of expression of HSV-TK in combination with ganciclovir treatment on cell proliferation. Cells of these lines infected ACNTK or AANTK or control virus ACN (Wills et al.,1994 supra), which directed the expression of HSV-TK, and then was treated with increasing doses of ganciclovir. The effectiveness of this treatment was a function of the concentration of ganciclovir. Determined this the end of the tap line was defined as the relative number of cells, in which expressibility transferred adenovirus genes, to cells, in which expressively marker gene of the bat-galactosidase, transferred control virus. The data presented in Fig. 14 and table 2, show that combined treatment with virus ACNTK and ganciclovir are able to inhibit cell proliferation of all tested lines (control in this case served as a treatment control adenovirus ACN in combination with ganciclovir). On the contrary, the viral vector AANTK was effective in those hepatocellular lines that expressed AFP. In addition, the combination AANTK/ganciclovir was more effective at inoculation of cells with high density.

"Nude" mice with tumors induced Hep3B cells (a group of five animals) were intratumorally or peritumorally equivalent dose ACNTK or control virus ACN. 24 hours after the first injection of recombinant adenovirus began daily administration of ganciclovir each mouse. Twice a week was determined using Vernier caliper sizes of the tumors in each animal and the average size of the tumors is shown in Fig. 16. On the 58th day the average size of tumors was lower in the treated ACNTK mice, the m effect ACNTK on tumor growth in vivo. Significant differences in average body weight between the groups of animals were not found.

Although the invention is described in relation to the above embodiments, it is necessary to understand that there are various modifications do not contradict the idea of the invention. Accordingly, the scope of the claims of the present invention is limited to the following formula.

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1. A pharmaceutical composition comprising a recombinant adenoviral expression vector, which contains (a) the insertion of exogenous DNA that contains a gene that encodes a foreign protein and (b) adenoviral DNA having a deletion starting at position from 357 to 360 and ending in position from 4020 to 4050.

2. The pharmaceutical composition under item 1, characterized in that it contains a deletion of nonessential DNA sequences in early region 3 and/or early region 4 adenoviral sequence.

3. The pharmaceutical composition according to p. 1, wherein the recombinant adenoviral expression vector contains delty adenoviral expression vector contains alien DNA molecule, encoding a polyadenylation signal.

5. The pharmaceutical composition according to paragraphs.1 to 4, characterized in that the adenovirus belongs to the group and selected from serotypes 1, 2, 5, or 6.

6. The pharmaceutical composition under item 1, characterized in that gene that encodes a foreign protein, is a DNA molecule size to 2.6 kilobase.

7. The pharmaceutical composition under item 1, characterized in that gene that encodes a foreign protein, is a DNA molecule size to 4.5 kilobase.

8. The pharmaceutical composition under item 1, characterized in that the gene encodes alien functional protein or biologically active fragment.

9. The pharmaceutical composition according to p. 8, characterized in that the gene encodes functional protein alien suppression of tumor or its biologically active fragment.

10. The pharmaceutical composition according to p. 1, wherein the suicide gene encodes a protein or its functional equivalent.

11. Method of gene therapy, characterized by administration to a mammal a pharmaceutical composition containing a recombinant adenoviral expression vector, which contains (a) the insertion of exogenous DNA containing GE is cancelaura in position from 4020 to 4050, and one or more pharmaceutically acceptable carrier.

12. The method of transformation of hyperproliferative mammalian cell, characterized in that the cells are contacted with a pharmaceutical composition comprising a recombinant adenoviral expression vector, which contains (a) the insertion of exogenous DNA that contains a gene that encodes a foreign protein and (b) adenoviral DNA having a deletion starting at position from 357 to 360 and ending in position from 4020 to 4050, and one or more pharmaceutically acceptable carrier.

13. The method of treatment of cancer, characterized by the introduction of pharmaceutical compositions containing recombinant adenoviral expression vector, which contains (a) the insertion of exogenous DNA that contains a gene that encodes a foreign protein and (b) adenoviral DNA having a deletion starting at position from 357 to 360 and ending in position from 4020 to 4050, and one or more pharmaceutically acceptable carrier.

14. The method according to p. 13, characterized in that the encoded gene functional alien protein is a protein suppression of tumors, and cancer is associated with loss of endogenous protein suppression of tumors in wild-type.

16. Method of inhibiting proliferation of a tumor in animals, characterized by the introduction of animal pharmaceutical compositions containing recombinant adenoviral expression vector, which contains (a) the insertion of exogenous DNA containing the gene encoding suicidal protein or its functional equivalent, and (b) adenoviral DNA having a deletion starting at position from 357 to 360 and ending in position from 4020 to 4050, and one or more pharmaceutically acceptable carrier.

17. A method of reducing proliferation of tumor cells, characterized by the introduction of the animal a pharmaceutical composition containing an effective amount of the metabolite timedancing or its functional equivalent, recombinant adenoviral expression vector, which contains (a) the insertion of exogenous DNA containing the gene encoding suicide is t 357 to 360 and ending in position from 4020 to 4050, and one or more pharmaceutically acceptable carrier.

18. The method according to p. 17, wherein the metabolite timedancing represents ganciclovir, or 6-methoxypurine arabinopyranoside, or their functional equivalent.

19. The method according to p. 18, wherein the tumor cells are the hepatocellular carcinoma.

20. Set to reduce proliferation of tumor cells, including recombinant adenoviral expression vector, which contains (a) the insertion of exogenous DNA containing the gene encoding suicidal protein or its functional equivalent, and (b) adenoviral DNA having a deletion starting at position from 357 to 360 and ending in position from 4020 to 4050, metabolite timedancing or its functional equivalent, one or more pharmaceutically acceptable carrier.

Priority points:

25.10.93 - PP.1 - 9, 11 - 15;

19.05.94 - PP.10, 16 - 20.

 

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The invention relates to medicine, namely to the use of inhibitors of proliferation of stem cells

The invention relates to medicine, namely to immunology and immunology, and concerns the problem of vaccination against tumor cells and vaccine therapy of cancer

The invention relates to medicine, in particular to the use of suppressor genes in malignant tumors, in combination with a DNA damaging agent or factor, with the aim of killing cells, in particular cells of malignant tumors

The invention relates to a vaccine for combating infections caused by In

FIELD: biotechnology, veterinary science.

SUBSTANCE: invention relates to therapeutic vector used in therapy of infectious diseases in cats that comprises at least one foreign nucleic acid each of that (a) encodes protein taken among the group consisting of feline protein CD28 represented in SEQ ID NO:8 or its immunogenic moiety; feline protein CD80 represented in SEQ ID NO:2 or 3, or its immunogenic moiety; feline protein CD86 represented in SEQ ID NO:6 or its immunogenic moiety, or feline protein CTLA-4 represented in SEQ ID NO:10 or its immunogenic moiety; and (b) nucleic acid that is able to be expressed in insertion of vector in the corresponding host. Indicated therapeutic vector is used in effective dose as component of vaccine against infectious diseases in cats for their immunization and in methods for enhancement or inhibition of immune response in cats and reducing or eradication of tumor in cats. Invention provides stimulating the activation and proliferation of T cells and to enhance effectiveness of control of infectious diseases in cats.

EFFECT: valuable biological properties of recombinant virus.

41 cl, 13 dwg

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