RU2194755C2 - Recombinant plasmid dna pad5-f carrying of adenovirus type 5 genome fragment with deletion in gene e1b-55k and strain of mutant adenovirus ade 12 exhibiting selective antitumor activity - Google Patents

Abstract

FIELD: biotechnology, medicine, oncology, genetic engineering, molecular biology. SUBSTANCE: invention presents recombinant plasmid DNA pAd5-f and mutant strain of human adenovirus Ade 12. Recombinant plasmid DNA carries fragment of genome of adenovirus type 5 with deletion in gene E1B- 55K. Mutant strain of adenovirus obtained by homologous recombination using plasmid pAs5-f exhibits the selective antitumor activity. Strain can be used as a base for the development of effective antitumor preparation. EFFECT: valuable medicinal properties of strain and plasmid. 2 cl, 3 dwg, 2 tbl, 4 ex

Description

 The invention relates to medicine and biotechnology, specifically to genetic engineering, and is a recombinant plasmid carrying a fragment of the type 5 adenovirus genome with a deletion in the E1B 55K gene and obtained by homologous recombination using this plasmid, the mutant human Aden2 virus adenovirus with selective antitumor activity .

 Recent studies have shown that cells of various types of human malignant tumors contain deletions or mutations in the p53 gene, which are accompanied by the loss of some functions of the protein encoded by them [1]. About 50% of human tumors are p53 defective, including malignant tumors of the lungs (60%), intestines (50%), breast (40%), ovaries (60%), head and neck (60%) [2]. Mutations or deletions of the gene encoding p53 represent one of the main reasons that determines the resistance of tumor cells to standard therapeutic effects [3]. The tumor suppressor gene p53 is one of the main factors that impede the growth of pathological cells in the body. In response to various injuries threatening genetically transmitted changes in the genome (mutagenic effects, activation of oncogenes, hypoxia, etc.), p53 is activated, as a result of which the cell stops dividing or dies by apoptosis [4, 5]. However, a number of DNA-containing viruses, such as adenoviruses, SV40 and human papillomavirus, encode proteins that inactivate p53 and thereby allow them to efficiently replicate in normal cells. Inactivation of p53 ensures cell viability during the reproduction of these viruses [4, 6, 8]. The E1B region of the adenoviral genome encodes the E1B 55K protein, which binds and inactivates p53. It is assumed that mutant variants of the virus that do not express the functional E1B 55K protein will be unable to efficiently replicate in normal human cells, but will retain replicative activity in tumor cells defective in the p53 protein gene and accordingly lyse them.

 Currently, various variants of recombinant adenovirus strains are described in foreign literature, such as with deletions in different parts of the genome, including the mutant variant of adenovirus dl1520, carrying a deletion of 827 bp. in the E1B region of the 55K gene [7, 9 - prototype], and with genes cloned in the genomic DNA encoding recombinant proteins with antitumor activity [10, 11]. In the works of J. R. Bischoff et al. and C. Heise et al. [9, 12] it was shown that the mutant variant of the adenovirus dl1520 (also known as ONYX-015) is capable of selectively infecting and lysing p53-defective human tumor cells in vitro, and also has oncolytic activity in intratumoral and intravenous administration to nude / nu mice with vaccinated human tumors. Currently, the USA is conducting clinical trials of this strain on patients with various forms of malignant neoplasms. However, in later studies of other authors [13, 14], carried out with the mutant strain dl1520, results were obtained that contradict J.R. Bischoff and C. Heise. In these studies, a panel of cell cultures, including both p53-defective and p53-positive human tumor cells and normal primary human cell lines, showed that the level of replicative activity of dl1520 in vitro does not correlate with the p53 status of the cells used. Moreover, in the work of Rothmann, T. et al. [14] it is noted that the mutant strain of adenovirus dl1520 infects and lyses normal human primary cells (MEC - epithelial cells of the breast, FF105 - foreskin fibroblasts) with an efficiency similar to that of the wild type of adenovirus. Thus, despite the fact that dl1520 (ONYX-015) is currently used in clinical practice (phase 1 of clinical trials), there are scientifically based doubts about its ability to selectively infect and lysis of p53-defective human tumor cells.

 An object of the invention is to obtain a mutant variant of adenovirus carrying a deletion in the E1B 55K gene, selectively replicating in p53-defective tumor cells and having oncolytic activity.

 The problem is solved by constructing recombinant plasmid DNA pAd5-f, carrying 3505 bp 5'-region of adenovirus genomic DNA with a deletion of 1118 bp in the E1B 55K gene, which is used to obtain the mutant variant of Adel2 adenovirus by homologous recombination.

The recombinant plasmid pAd5-f is characterized by the following features:
- has a length of 6231 bp and a molecular weight of 4.05 kDa;
- contains a vector fragment of EcoRI-XbaI from plasmid pRS-2 with a size of 2726 bp

- carries a fragment of EcoRI-XbaI 5'-region of genomic DNA of type 5 adenovirus with a size of 3505 bp with a deletion after the translation termination site of the E1B19K gene of 1118 bp containing the SalI restriction endonuclease site, immediately after which the translation termination site of the E1B 55K gene is introduced;
- carries the following genetic markers: ampicillin resistance gene;
- unique restriction endonucleases sites having the following coordinates:
SalI - 1604
EcoRI - 3945
Kpn2.1 - 3114
XhoI - 430
The method for constructing plasmid pAd5-f consists in embedding amplification products obtained using the calculated primers and type 5 adenoviral DNA matrix into plasmid pRS-2 (derived from pUC18 with unique unique sites in the polylinker) at the corresponding polylinker sites.

 The design scheme is described in example 1 and illustrated in figure 1. To create a mutant variant of adenovirus, experiments were conducted on co-transfection of the plasmid pAd5-f linearized at the EcoRI site and adenovirus DNA treated with ClaI restriction endonuclease and alkaline phosphatase CIAP. Transfection was carried out by calcium-phosphate precipitation method. Mutant strains were selected using PCR with specific primers. The Adel2 amplification product was verified by restriction mapping and partial sequencing of the region adjacent to the deletion site.

 The essence of the invention lies in the fact that using the target plasmid DNA pAd5-f, carrying a fragment of the 5'-region of genomic DNA of type 5 adenovirus size 3505 bp with a deletion in the E1B 55K gene, 1118 bp in size after the translation termination signal of the E1B 19K gene, which has a SalI restriction endonuclease site immediately after which the translation termination signal of the E1B 55K gene is introduced, a mutant strain of adenovirus Adel2 with specific antitumor activity for p53-defective tumor cells is obtained by homologous recombination.

The obtained mutant variant of Adel2 type adenovirus 5 differs from the prototype of the mutant adenovirus dl1520 by the length of the deletion in the E1B 55K gene (in the prototype 827 bp, in the claimed virus 1118 bp), as well as by the presence of a unique SalI restriction site, which allows its use in further, for the insertion of specific nucleotide sequences that enhance the selectivity of this mutant variant. The resulting strain of Adel2 is characterized by the following features:
Morphological signs.

 The mutant variant Adel2 possesses all the typical morphological properties of type 5 adenoviruses.

 Physico-biochemical characteristics.

 The DNA of strain Adel2 has a length of 34817 bp (the wild type of adenovirus of the 5th serotype is 35935 bp), while the open reading frame of the E1B 55K gene is violated, the structural proteins do not differ from the proteins of the wild-type virus.

 Pathogenicity for animals.

 A study of the biological properties of strain Adel2 in animals showed that the strain is not pathogenic for laboratory animals. In standard tests for toxicity, safety and specificity, the recombinant strain does not differ from the original strain of type 5 adenovirus (Ad5).

 Replicative properties of the strain.

 The mutant Adel2 variant, carrying a deletion of the E1B 55K gene, has a high degree of replicative activity for complementary cells of 293 and p53-defective tumor cells (A431, SW480, and HEP2) and, at the same time, has significant replication restrictions in normal fibroblasts of adult skin (HSF) and human embryonic fibroblasts (HSEF) (Table 1).

 Oncolytic activity of the strain.

 Strain Adel2 has a pronounced inhibitory effect on the development of nu / nu grafted mice in human tumors (A431 cell line). The strain of the mutant adenovirus Adel2 was deposited at the Research Institute of KKM SSC VB "Vector" under the number V-336.

The invention is illustrated by the following graphic images:
FIG. 1 is a schematic of the recombinant plasmid pAd5-f and a method for constructing it.

 Figure 2 - PCR analysis of the mutant adenovirus Adel2 DNA, where: M - markers, 1 - plasmid pAd5-f, 2 - cell culture control, 3 - Adel2, 4, 5, 6 - wild type 5 adenovirus strain.

 FIG. 3 - reproduction level of the mutant Adel2 virus in p53-defective tumor and normal cells, where A431, SW480 and Hep2 - p53-defective human tumor cells; 293 - human kidney embryonic cells transformed with E1A and E1B genes of adenovirus; HSF - human skin fibroblasts; HSEF - embryonic fibroblasts of human skin.

 The invention is illustrated by the following examples.

 Example 1. Construction of plasmid DNA pAd5-f.

 Procedures for treating plasmid DNA with restriction endonucleases, modification enzymes, electrophoretic separation of DNA hydrolysates, polymerase chain reaction, isolation of DNA fragments from agarose gel, ligase reactions, transfection of eukaryotic cells, isolation of plasmid DNA, sequencing according to the Sanger method are carried out in accordance with standard genetic engineers methods.

 Specific oligonucleotides 603, 609, 610, 608, A, F are calculated according to the Ad5 sequence using the Oligo 3.0 program.

Oligonucleotide sequences:
603 5'-CGGAATTCGAGTGCCAGCGAGTAGAGTTTT-3 '
609 5'-ACGCGTCGACCTCCCTCTTTTACCCCCTTTAGC-3 '
610 5'-ACGCGTCGACGTGATGCTGGATGTGACCGA-3 '
608 5'-CGCGGATCCACAAGGGGCGTCTCCAAGTT-3
A 5'-GCCAGCCTCGTGGCAGGTAAGATCG-3 '
F 5'-CGGAATTCATCATCAATAATATACCTTAТТТТTGG-3 '
Isolation of genomic DNA of type 5 adenovirus for polymerase chain reaction is carried out as follows.

 At the first stage, the production of wild type human adenovirus of the 5th serotype (Ad5) is carried out on a subconfluent monolayer culture of 293 cells (human embryonic kidney cells transformed with Ad5 E1A and E1B genes). Upon reaching 70% of the cytopathogenic effect of the virus on the cell culture, the cells are selected and concentrated by centrifugation (1500 g, 5 min). The homogenate of infected cells is extracted with an equal volume of carbon fluoride, then the aqueous fraction containing the virus is centrifuged on a gradient of cesium chloride (1 / 45-1 / 33 g / ml, 2 hours at 90,000 g). To free the virus from residual amounts of cellular components, additional purification is carried out on a preformed gradient of cesium chloride (1 / 45-1 / 33 g / ml, 6 hours at 100,000 g). The virus-containing fraction was selected and diluted in a 1: 4 ratio with Tris-EDTA buffer and high-speed centrifugation was performed to precipitate the virus, after which the precipitate was resuspended in 3 ml of Tris-EDTA buffer (5 mM Tris-HCl, 1 mM EDTA, pH 7, 8).

In a second step, viral DNA is isolated from the purified virus. For this, proteinase-K is added to the viral preparation to a concentration of 20 μg / ml and sodium lauryl sarcosylate to a concentration of 0.5%, incubated at 37 ^{° C.} for 3 hours. Then an equal volume of phenol saturated with Tris-HCl buffer is added to the tube; pH 8.0. The solution was stirred and centrifuged 2000 g, 10 min. The upper aqueous phase is transferred to another tube, DNA is precipitated by the addition of 2 ^x volumes of ethanol, followed by centrifugation of 2000 g, 10 min. The precipitate was dissolved in 100 μl of distilled water.

For polymerase chain reaction using genomic DNA of type 5 adenovirus isolated by the phenolic method. The following temperature regimes are applied from 1 to 36 cycles: 95 ^o C - 20 s, 50 ^o C - 20 s, 72 ^o C - 30 s, 37 cycle 95 ^o C - 20 s, 50 ^o C - 20 s, 72 ^o C - 7 minutes Amplification product (10 μg) 603/609 1827 bp treated with restriction endonucleases EcoRI-SalI ("SibEnzyme"). 2 μg of plasmid DNA pRS-2 is treated with EcoRI-SalI restriction enzymes. Aliquots of the PCR fragment and plasmid DNA are used for ligase reaction in a volume of 20 μl. 10 μl of ligase mixture is used to transform competent E. coli JM103 cells. Transformants are scattered on LB agar containing 100 μg / ml ampicillin. The grown clones are used to generate and isolate recombinant plasmid DNA. Plasmid DNA is then subjected to restriction mapping and clones carrying the target insert of 1827 bp (pAd1), produce, isolated and used as a vector for cloning the second amplification product 610/608. Amplified product 610/608 1157 bp (10 μg) are treated with restriction endonucleases SalI-BamHI. Plasmid DNA pAd1 (2 μg) is treated with restriction enzymes SalI-BglI, then the resulting hydrolyzate is subjected to dephosphorylation using alkaline phosphatase, vector plasmid DNA is isolated. Aliquots of the PCR fragment and vector DNA are used to conduct the ligation reaction in a volume of 20 μl, and 10 μl of the ligase mixture is taken for transformation of competent cells. Clones bearing target insert 2984 bp 5'-regions of adenoviral DNA (pAd5) are generated and used as a vector for insertion of a 932 bp AF fragment of PCR amplification. 2 μg of plasmid DNA pAd5 is treated with restriction endonucleases EcoRI-Kpn2,1; the resulting hydrolyzate is separated by electrophoresis in 1% agarose gel; isolated the vector part of the plasmid with a length of 5421 bp 10 μg of the PCR amplification product AF is treated with restriction endonucleases EcoRI-Kpn2,1; the resulting hydrolyzate is separated by electrophoresis in 1.5% agarose gel; a fragment of 827 bp is isolated Aliquots of the PCR fragment and vector DNA are used to conduct a ligation reaction in a volume of 20 μl, and 10 μl of the ligase mixture is taken for transformation of competent E. coli JM103 cells. Transformants are plated on LB agar containing 100 μg / ml ampicillin. The grown clones are used to generate and isolate recombinant plasmid DNA, which is analyzed by restriction mapping and further clones carrying the target insert 3505 bp produce for detailed restriction mapping and sequencing by the Sanger method. The target plasmid pAd5-f includes a fragment of the 5'-region of the genomic DNA of type 5 adenovirus with a length of 3505 bp with a deletion in the region of the E1B 55K gene and contains the unique SalI restriction site. Scheme of plasmid DNA pAd5-f and the method of its construction are presented in figure 1.

 The constructed recombinant plasmid DNA pAd5-f is used for homologous recombination with adenoviral DNA and to obtain a mutant variant of the virus with deletion in the E1B 55K gene.

 Example 2. Obtaining mutant adenovirus Adel2.

 For transfection of cell cultures, Ad5 virus DNA is used, which is in complex with the terminal protein, which is obtained from the purified viral preparation by the guanidine-chloride method. For this, an equal volume of 8 M guanidine chloride is added to the viral suspension, the solution is stirred and applied to a chromatographic column with Sepharose-4B, equilibrated with 4 M guanidine chloride. DNA leaving the first peak in optical density is collected, dialyzed against 0.01 M Tris-Hcl buffer containing 0.001 M EDTA, poured into aliquots, frozen and stored in liquid nitrogen.

For cotransfection, 10 μg of pAd5-f plasmid is treated with EcoRI restriction endonucleases, 15 μg of type 5 adenovirus DNA is treated with ClaI restriction endonucleases and alkaline phosphatase modification enzyme CIAP. Kubariki with a monolayer of a transplantable cell line of a human embryo kidney transformed with E1A and E1B Ad5 genes are washed with phosphate buffer and co-transfected with 1 μg of viral DNA in combination with the terminal protein mixed with a 10-fold excess of plasmid DNA using standard calcium phosphate transfection technique. To 450 μl of a mixture of linearized plasmid DNA pAd5-f and adenovirus DNA in sterile deionized water add 50 μl of 2.5 M CaCl ₂ . The resulting DNA / CaCl ₂ mixture was added to 500 μl of HEPES buffer and allowed to stand for 20 minutes to form a precipitate. Apply to the cell monolayer precipitate and serum-free DMEM. After 24 hours, the medium is removed and normal DMEM growth medium with gentamicin and 10% FCS is added. Five days after transfection, a cytopathogenic effect on cell culture is detected in the case of transfection of native DNA and in cases of transfection of cells with EcoRI-treated adenoviral DNA together with the linearized plasmid pAd5-f. To preliminarily confirm the presence of target mutations in the genome of the resulting transfected viruses, PCR analysis is performed. As a matrix, viral DNA is used, isolated from preparations of the obtained variants of adenovirus as a result of triple cloning by the plaque method. When conducting PCR, primers 605/608 are used.

Primer Sequences
605: 5'-TCTGAGCGGGGGGTACCTGCTGGATT-3 '
608: 5'-CGCGGATCCACAAGGGCCTCTCCCAAGTT-3
As a result of PCR, the product of 1422 bp is amplified. (in control with a wild strain, the PCR product is 2540 bp). The results of PCR analysis, confirming the presence of a target deletion in the E1B 55K gene, are presented in Figure 2.

 The preparative production of a cloned mutant Adel2 variant through a plaque was performed on 293 cell culture. The resulting virus preparation was purified in the gradient of cesium chloride, after which the corresponding viral DNA was isolated by the phenol method. At the next stage, the polymerase chain reaction using the Adel2 variant as a matrix of genomic DNA amplifies a fragment of the genome corresponding to E1B 55K (2048-4582 bp). After that, the amplified fragments are sequenced according to the Sanger method.

 Determination of the nucleotide sequence of the E1B 55K gene of the mutant variant Adel2 shows a deletion of 1118 bp (the length of the intact gene is 1488 bp) after the translation termination signal of the E1B 19K gene. In addition, there is a site for SalI restriction endonuclease introduced during the construction of the mutant strain Adel2. Directly behind the SalI site is a translation termination signal for the E1B 55K gene.

 Example 3. Characterization of the replicative properties of Adel2 on p53-defective tumor cells and normal human fibroblasts.

 Dulbecco modified Eagle medium (DMEM) and fetal serum (FCS) manufactured by Life Technology (Gibco BRL, Germany) were used to cultivate cell cultures and produce viral strains. 293, HEp2, SW480, and A431 cells (p53-defective human tumor cells) were cultured as monolayer cultures on DMEM medium containing 10% FCS with 80 μg / ml gentamicin sulfate. Normal primary human skin fibroblasts and human embryo skin fibroblasts (HSF and HSEF, respectively) are cultured as monolayer cultures on DMEM containing 10% FCS with 80 μg / ml gentamicin sulfate.

After additional cloning of the mutant variant of Adel2 and confirmation by the method of polymerase chain reaction of the absence of wild-type adenovirus impurities in Adel2 preparations, the infectivity of the mutant variant for normal human fibroblasts and various p53-defective tumor cells is studied in comparison with the wild type of adenovirus. The infectivity of Adel2 and wild-type adenovirus is assessed by the cytolytic effect on cell cultures and their reproductive activity on the panel of the studied cell cultures. A study of the cytolytic activity of the mutant variant and wild-type adenovirus on a panel of p53-defective tumor and normal human cells is carried out using a micromethod according to Chanas et al. on 96-well culture plates (Costar); the viral infectious titer for the respective cell types is expressed in terms of TCP ₅₀ (50% tissue cytopathogenic dose). Before the experiment, Adel2 and wild-type adenovirus preparations were titrated on 293 cells and resulted in one titer. The results are presented in table 1. To determine the reproductive properties of viruses, normal fibroblasts and p53-defective human tumor cells infect Adel2 or wild-type adenovirus with a multiplicity of 1 PFU / cell. 48 hours after infection, the corresponding virus is released from the cells by triple freezing; supernatants obtained after centrifugation are titrated on 293 cells. The amount of Adel2 produced 48 hours after infection was normalized against the amount of wild-type adenovirus produced in the same cell line over the same period of time. The results are shown in FIG. 3. A mutant variant of Adel2, carrying a deletion of the E1B 55K gene, infects complementary 293 cells and p53-defective tumor cells (A431, SW480 and HEP2) with the efficacy of wild-type adenovirus. At the same time, it has significant limitations on replication in normal p53-positive fibroblasts of adult skin and embryonic fibroblasts of human skin in comparison with the wild type of adenovirus.

Example 4. The study of the oncolytic activity of the mutant strain Adel2
To study the oncolytic efficacy of the mutant variant Adel2 in experiments in vivo, its preparative life is carried out with subsequent purification in the density gradient of cesium chloride. The resulting preparation is titrated on a 293 cell culture, infectious titers must be at least 5 • 10 ¹⁰ PFU / ml.

The study of oncolytic activity is carried out on 8-week nude / nu nude mice that have undergone a preliminary two-week quarantine and kept under aseptic conditions. For vaccination of animals using a culture of human tumor cells A431. According to published data, these human tumor cells effectively form tumors by subcutaneous administration of nu / nu to mice. 1.0 x 10 ⁷ cells in 0.2 ml of DMEM are injected subcutaneously in each flank of nu / nu mice. Tumor volumes are determined according to the following formula: (max) • (perpendicular width) ^2/2. Vaccination of A431 cells in mice typically results in progressive tumor growth in all animals. Upon reaching the volume of A431 tumors, which allows intratumoral administration, the tumors are treated with Adel2. For this purpose, 10 ⁹ PFU of the study drug is injected into each tumor in 60 μl of DMEM (15 μl in each quadrant of the tumor). Injections are carried out daily for 5 days. Control tumors (vaccinated with control animals) are treated according to the same scheme with Adel2 inactivated with ultraviolet radiation. The results are presented in table.2. They indicate that the treatment of tumors with Adel2 leads to a pronounced inhibition of tumor cell growth both in average values of tumor volumes and in the multiplicity of increase in volume from the start of observation to its completion.

 Thus, a recombinant plasmid DNA pAd5-f was constructed that carries a fragment of the type 5 adenovirus genome with a deletion in the E1B 55K gene, and a mutant strain of human adenovirus Adel2 with selective antitumor activity was obtained by homologous recombination.

LITERATURE
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Claims (2)

 1. Recombinant plasmid DNA pAd5-f carrying a fragment of the genome of type 5 adenovirus with a deletion in the E1B-55K gene, having a molecular weight of 4.05kDa, containing EcoRI-XbaI vector fragment of plasmid pRS-2, EcoRI-XbaI fragment 5 '- genomic region Type 5 adenovirus DNA with a deletion after the translation termination site of the E 1B 19K gene of 1118 bp containing an endonuclease site, immediately after which the translation termination site of the E 1B-55K gene is introduced; ampicillin resistance gene as a genetic marker; unique sites of restriction endonucleases SalI, EcoRI, Kpn2.1, XhoI.  2. The strain of adenovirus type 5 Ade12 V-336, obtained using recombinant plasmid DNA pAd5-f according to claim 1, with selective antitumor activity.

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