Oncolytic adenovirus for treating cancer, using it, and pharmaceutical composition containing it

FIELD: medicine, pharmaceutics.

SUBSTANCE: presented inventions refers to biotechnology, and particularly to oncology and concerns an oncolytic adenovirus, using it and a pharmaceutical composition containing this adenovirus. The characterized adenovirus contains a sequence intervening into its genome and coding hyaluronidase. The enzyme expression is controlled by a promoter active in animal's cells. The presented adenovirus can be used for preparing an agent for treating cancer and pre-cancer.

EFFECT: invention enables providing higher effectiveness and selectiveness of the therapy.

20 cl, 11 dwg, 10 ex

 

The invention relates to medicine, in particular to the field of Oncology, namely virotherapy.

BACKGROUND of the INVENTION

Currently, the treatment of cancer is based primarily on chemotherapy, radiation therapy and surgery. Despite the high degree of success in the treatment of cancer in the early stages, the most advanced cases of cancer are incurable, because they cannot be removed surgically or because the dose radio - or chemotherapeutic substances is limited due to their toxicity towards normal cells. To mitigate this situation have been developed biotechnological approaches to improve the efficacy and selectivity of cancer treatment. Among them, gene therapy and virotherapy use viruses for therapeutic purposes against cancer. In gene therapy virus modify to prevent its replication and for use as delivery vehicles or for use as a vector for therapeutic genetic material. On the contrary, in virotherapy use a virus that replicates and replicates selectively in tumor cells. If vinotherapie tumor cells die due to the cytopathic effect caused by replication of the virus inside them, but not at the expense of therapeutic is Jena. Preferential replication in cancer cells is called ecotropica, and lysis of tumor is called oncletom. In the strict sense, the viruses that selectively replicate in tumors, called oncolytic, although in a broader sense, the word "oncolytic" can be applied to any replication-competent virus is able to lyse tumor cells, even without selectivity. In this description, the term "oncolytic" is used in both senses.

Virotherapy cancer is preceded by gene therapy. First studies for the treatment of tumors viruses belong to the beginning of the last century. In 1912 De Pace achieved regression of tumors after injection of rabies virus in cervical carcinoma. Since then, the tumor for treatment have introduced many kinds of viruses. There are viruses that display natural oncotropic, such as Autonomous parvovirus, vesicular stomatitis virus and reovirus. Other viruses for selective replication in tumors can be manipulated genetically. For example, herpes simplex virus (HSV) is onkotrone in the elimination of the gene ribonucleotides not necessary enzymatic activity in actively proliferating cells such as tumor cells. While adenovirus, due to its low pathogenicity and high abilities infection is SQL tumor cells is the virus most often used in virotherapy and gene therapy of cancer.

Fifty-one serotype of adenovirus person was identified and classified into 6 different groups from A to F.

The human adenovirus type 5 (Ad5), which belongs to the group C is a virus, educated protein the icosahedral capsid, which is Packed linear DNA length 36 thousand pairs of nucleotides. In adults Ad5 infection is usually asymptomatic, but in children it causes a runny nose and conjunctivitis. Usually Ad5 infects epithelial cells during natural infection are the cells of the bronchial epithelium. He enters the cell through the interaction of a fiber, a viral protein which is extracted as the antenna of the twelve vertices of the capsid, with cellular proteins involved in intercellular adhesion, which is called adenovirus receptor Coxsackie (CAR). When you hit the viral DNA into the nucleus begins ordered the transcription of early genes (E1 through E4) virus. The first expressed viral genes are genes early region 1A (EA). EA associated with cellular protein Rb for the release of E2F, which activates the transcription of other viral genes, such as E2, E3 and E4, and cellular genes that activate the cell cycle. On the other hand, EV associated with p53 to activate kletocnah the cycle and prevent apoptosis of infected cells. E2 encodes proteins involved in virus replication; E3 encodes proteins that suppress antiviral immune response; E4 encodes proteins involved in the transport of viral RNA. Expression of early genes leads to replication of viral DNA, and after the DNA is replicated, activates the main late promoter and initiates transcription of messenger RNA, which after differential splicing forms all RNA encoding the structural proteins that form the capsid.

There are two important to consider aspect in relation to the development of oncolytic adenoviruses: selectivity and activity. In order to achieve selectivity against tumor cells, were used three strategies: elimination of viral functions necessary for replication in normal cells but are not required in the tumor cells; the control of viral genes that trigger replication using promoters that are selectively active in tumors; and modification of the capsid proteins of the virus are important in the infection of a host cell. Such genetic modifications can provide a significant level of selectivity, with the efficiency of replication in tumor cells, 10,000 times greater than the efficiency of replication in normal cells. Against oncolytic activity for its increase was described several genetic modifications. These changes include: a) increasing the release of viruses, for example, by elimination E1B19K, superexpression E3-11.6 K (ADP) or localization of the protein E3/19K in the plasma membrane; and (b) integration of therapeutic gene into the genome of oncolytic adenovirus for education "charged oncolytic adenovirus". In this case, therapeutic gene could mediate the death of uninfected tumor cells through activation of prodrugs with Supervisory action (that is to say, that destroys uninfected neighboring cells), activation of the immune system against tumors, induction of apoptosis, inhibition of angiogenesis or elimination of extracellular matrix, among others. In these cases, the method and time of expression of therapeutic gene will be crucial for the final outcome of therapeutic approach.

In the last decade, patients with carcinomas of the head and neck, ovarian, colon and rectum, pancreas and malignant hepatoma, among others, have introduced a variety of oncolytic adenoviruses. The safety profile of these adenoviruses in clinical research has been very promising. Discovered side effects such as flu-like symptoms and increased levels of transaminases were well tolerated even after systemic injections of high doses of virus (see D. what about the et al., "Development of transcriptionally regulated oncolytic 25 adenoviruses",Oncogene2005, vol. 24, pp. 7763-74; and T. Reid et al., "Intravascular adenoviral agents in cancer patients: lessons from clinical trials",Cancer Gene Therapy2002, vol. 9, pp. 979-86). Although the introduction of recombinant adenovirus induced partial inhibition of tumor growth, the complete eradication of tumors has not been reached, and after a short period of time the tumor again rapidly. These results probably occurred because the entered adenovirus was distributed only on a small part of the tumor, producing limited antitumor response, as uninfected cells continued to grow rapidly. In a recent study it was noted that replication of oncolytic adenoviruses in xenotransplantation human tumors lasted from 35 to 100 days after systemic administration, although it does not lead to complete eradication of the tumor (see H. Sauthoff et al., "intratumoural spread of wild-type adenovirus is limited to after local injection of human xenograft tumours: virus persists and spreads systemically at late time points,Human Gene Therapy2003, vol. 14, pp. 425-33). This low antitumor efficacy is partly because the connective tissue and extracellular matrix (ECM) in the tumor prevents the spread of adenovirus inside the tumor.

This effective dissemination of oncolytic adenoviruses in the tumor weight was also described for other protivoopujoleve who's funds, such as doxorubicin, Taxol, vincristine, or methotrexate. Many studies indicate the role of the ECM in the resistance of tumor cells to chemotherapeutic agents (see BP'toole et al., "Hyaluronan: a constitutive regulator of chemoresistance and malignancy in cancer cells",Seminars in Cancer Biology2008, vol. 18, pp. 244-50). Tumor and stromal cells produce and form a matrix of collagen, proteoglycans and other molecules, which hinders the transport of macromolecules inside the tumor. Hyaluronic acid (NA) is one of the main ECM components involved in the resistance of tumor cells to therapeutic drugs. ON sverkhekspressiya in a variety of malignant tissues, and in many cases the level is a factor in the prediction of progression of the tumor. Interaction with receptors CD44 and RHAMM increases the survival rate of the tumor and invasion. IN addition, can induce tumor metastases, causing cell adhesion and migration, and protection from the immune system.

On the other hand, inhibition of the interaction between hyaluronic acid and tumor cells returns resistant to multiple drugs. Various studies have shown that hyaluronidase (enzymes that cause degradation ON) enhance the effect of different chemotherapies in patients with melanoma, sarcoma, head and neck tumors, the metastasis of the mi liver and carcinoma of the colon. The mechanism of action of hyaluronidase still unknown, but, as a rule, it is connected with reduction of cell adhesion barriers, decrease interstitial pressure and improve the penetration of anticancer drug in the tumor and not to its inhibitory effect on the signaling pathway associated with cell survival.

Recently it was described that the joint introduction of hyaluronidase and oncolytic adenoviruses using intratumoral injection reduces the progression of tumors (see S. Ganesh et al., "Intratumoural coadministration of hyaluronidaseenzyme and oncolytic adenoviruses enhances virus potency inmestastasic tumour models",Clin Cancer Res2008, vol. 14, pp. 3933-41). In these studies oncolytic adenoviruses enter four intratumoral injections and hyaluronidase injected nutripure on all other days throughout the treatment. This mode of introduction is small patients, since most tumors are not available for intratumoral injection. The treatment proposed by Ganesh with co-workers may not benefit patients with diffuse disease (metastases).

Despite these efforts, currently still necessary to search for new therapeutic approaches that are effective in the treatment of cancer.

The INVENTION

The inventors have found that the adenovirus, which re is glaziruetsya and contains hyaluronidase gene in its genome, distributed more efficiently in tumor mass. Expression of hyaluronidase oncolytic adenovirus results in the degradation of hyaluronic acid, which is part of the extracellular matrix of the tumor. Degradation of hyaluronic acid leads to a decrease interstitial pressure in the tumor and less resistance of the tumor to the spread of adenovirus, and consequently, to improve the spread of the virus from cell to cell in tumor formation. This improved distribution embodied in enhancing oncolytic effect. The authors found that when intravenous injecting oncolytic adenovirus according to the invention is achieved regression of tumor volume. Thus, oncolytic adenovirus of the present invention is suitable for the treatment of cancer. In addition, the gene expression of hyaluronidase no impact on viral replication or cytotoxicity of oncolytic adenovirus.

As mentioned previously, it has been described that intratumoral co-administration of oncolytic adenovirus and soluble hyaluronidase enhances the antitumor efficacy of oncolytic adenovirus. However, until this invention hyaluronidase gene was not introduced in any oncolytic adenovirus for cancer treatment.

As described in the examples, intratumoralin vivo

In addition, the administered dose of the adenovirus according to the invention are smaller: Ganesh et al. (above) introduces four intratumoral injections of 1×1010viral particles, whereas in the present invention introduces a single intravenous dose of 2×109viral particles. This means a reduction in dose 20 times and the advantage that the dose is one-time. In our approach, Ganesh et al. injected hyaluronidase nutripure every other day throughout the experiment. In addition, adenovirus also introduce nutripure at the beginning of treatment. Such intratumoral injection of virus and hyaluronidase is hardly applicable in the clinic, because most tumors are n is available for intratumoral injection. Presumably, the introduction of joint solution with hyaluronidase and adenovirus was not done systematic way, since the probability that both components together achieved dispersed through the body of tumor cells is low.

The present invention makes possible the expression of hyaluronidase in area and in time, when there is replication of the virus. This expression of hyaluronidase enhances the spread of the virus in tumor mass and increases its antitumor activity. It is expedient to introduce the adapted dose, non-toxic to animals, with great effectiveness for treatment.

In the present invention, oncolytic adenoviruses achieve tumor target cells. Once inside, the virus has replicated, expressed his capsid proteins and, at the same time, is expressed hyaluronidase encoded by the genome of the adenovirus. This hyaluronidase has been modified for release in the extracellular environment of the cell. In the extracellular environment hyaluronidase breaks the matrix and helps adenoviruses, which replicated the infection of neighboring tumor cells.

Thus, the aspect of the invention relates to the oncolytic adenovirus that contains integrated in its genome sequence, encoding the enzyme hyaluronidase.

In the framework of the invention, the term "oncolytic adenovirus" refers to the adenovirus, which is able to replicate or which is competent for replication in cancer cells. In this description oncolytic adenovirus and adenovirus can replicate are synonyms. They differ from rereplacenocase adenovirus, because the latter is unable to replicate in the target cell. Nerealizirane are adenoviruses used in gene therapy as carriers of genes to target cells, since the goal is the expression of therapeutic gene in intact cells, but not lysis of the cells. Instead, therapeutic effect of oncolytic adenovirus based on the ability to replicate and lyse the target cell, in particular a tumor cell that you want to eliminate.

Another aspect of the invention relates to a pharmaceutical composition that contains a therapeutically effective amount of oncolytic adenovirus together with pharmaceutically acceptable carriers and excipients.

Another aspect of the invention relates to the oncolytic adenovirus according to the invention for its use as a medicine.

Another aspect of the invention relates to the oncolytic adenovirus according to the invention for the treatment of cancer and the and precancerous forms of cancer in a mammal, including humans.

Another aspect of the invention relates to the use of oncolytic adenovirus to obtain drugs for the treatment of cancer or precancerous forms of cancer in a mammal, including humans. Treatment is based on replication of these oncolytic adenoviruses in tumors. Alternatively, this aspect of the invention can be formulated as a method of treatment of a mammal, including humans, cancer or precancerous forms of cancer, which includes an introduction to the specified mammal an effective amount of oncolytic adenovirus.

Another aspect of the invention relates to a Shuttle vector that can recombine with the genome of adenovirus, for the design of oncolytic adenovirus according to the invention. This vector contains inverted terminal repeat sequences of adenovirus ("inverted terminal repeats", ITR), a sequence that promotes expression of the sequence encoding the enzyme hyaluronidase, the sequence that encodes the enzyme, and a polyadenylation sequence.

In the private embodiment, the oncolytic adenovirus according to the invention is a human adenovirus, meaning that infected people. In particular, the human adenovirus selected from the group consisting of serotypes 151 adenovirus person and their derivatives. Under "derivative" refers to a recombinant adenoviral hybrid of two or more of the adenoviruses of different serotypes, for example, adenovirus serotype 5 fiber of adenovirus serotype 3. In the private embodiment of the invention oncolytic adenovirus person belongs to serotype 5.

Hyaluronidase are a family of enzymes that degrade hyaluronic acid. In humans there are 6 genes encoding hyaluronidase with different properties and locations. Isoforms Hyal1 and Hyal2 are present in most tissues. Hyal1 is the predominant form in plasma. Hyal3 present in bone marrow and testes, but its function is poorly understood. Hyaluronidase PH20 highly expressed in testis and involved in the process of fertilization of an oocyte by a sperm. Hyaluronidase PH20 anchored in the plasma membrane and in the inner acrosomal membrane of the sperm and makes the sperm ability to penetrate the extracellular matrix accumulation (rich in hyaluronic acid) to achieve the transparent area of the oocyte. During acrosomal reaction part of hyaluronidase, anchored in the membrane of the sperm, enzymatically processed to form a soluble form of the protein that is released from acrosomal membrane. In addition, hyaluronic acid is Idasa was identified as a factor in the spread of the venom of snakes, spiders, Scorpions and wasps.

In the private embodiment, the enzyme hyaluronidase is a testicular hyaluronidase mammal, more specifically, testicular hyaluronidase human. Testicular hyaluronidase human (GenBank GenelD: 6677) also known as SPAM1 or molecule 1 adhesion of sperm, and as a PH-20. Membrane protein PH20 is the only enzyme family hyaluronidase mammal activity at neutral pH. Encoding gene's forms two transcription options: option 1 longer, which encodes the isoform 1 protein (inventory number GenBank NP_003108.2) and option 2, which uses the signal of alternative splicing of 3'-end coding region, compared to variant 1, resulting in the isoform 2 with a shortened C-end (inventory number GenBank NP_694859.1).

In the private embodiment of the invention the sequence of the enzyme deleteroute sequence corresponding to the carboxy-end of the membrane-binding domain, for producing a soluble enzyme (see FIG.2). Deletion of this carboxykinase domain leads to the secretion of hyaluronidase in the extracellular environment. Thus was obtained oncolytic adenovirus that expresses secreterial hyaluronidase with enzymatic activity at neutral pH. In the private embodiment of th is sequence, integrated into the genome of the adenovirus, is a sequence which encodes SEQ ID NO: 1. In yet another private embodiment, the built-in sequence represents SEQ ID NO: 2.

In another embodiment, the sequence of the enzyme is embedded in oncolytic adenovirus after nucleotide sequence of the fiber of adenovirus.

In another embodiment, the expression of the enzyme is controlled by a promoter active in the cells of animals. In particular, the promoter is chosen from the group consisting of a cytomegalovirus promoter, the chief of the late promoter of adenovirus, SV40 promoter, promoter timedancing of herpes simplex virus, RSV promoter, promoter EF1-alpha promoter beta-actin promoter, IL-2 person, promoter of IL-4, IFN promoter, E2F promoter and promoter GM-CSF person. The promoter that controls the expression of the enzyme can be a natural promoter of adenovirus, as occurs in the case of the main late promoter of adenovirus (see FIG.1(a), MLP, "the major late promoter"). The promoter can also be embedded before the sequence that encodes the enzyme. In a preferred embodiment, the promoter is the major late promoter of adenovirus.

Replicative adenovirus according to the invention may have a modification in St. what she genomic sequence, which give the ability of selective replication in tumor cells. In the private embodiment, this is achieved by embedding tissue-specific promoter or tumor-specific promoter. This promoter controls the expression of one or more genes of the group Ia, E1b, E2 and E4. In particular, the promoter is selected from the group consisting of E2F promoter, telomerase hTERT promoter, tyrosinase promoter, promoter specific antigen of the prostate (PSA), alpha-fetoprotein promoter, the promoter of COX-2, as well as synthetic promoters formed by multiple binding sites of transcription factors, such as the binding sites of factor induced by hypoxia (HIF-1), transcription factor Ets, tumor cytotoxic factor (tcf), the transcription factor E2F or the transcription factor Sp1. Preferably, the promoter controls the expression of EA.

Another modification to obtain selective replication in tumors is the elimination of the functions EA, which blocks retinoblastoma (RB) pathway. Other viral genes that interact directly with pRB, such as E4 and E4orf6/7, are candidates for elimination to achieve selective replication in tumor cells. As shown in the examples, the oncolytic adenovirus ICOVIR17 aracterized simultaneous content hyaluronidase gene, a deletion of the A24, which affects the interaction EA with pRB, including four E2F1-binding sites and one Sp1-binding site in the endogenous promoter EA to control the expression of EA and, finally, the inclusion of RGD-peptide in the fiber of adenovirus to increase infectious virus activity. ICOVIR17 represents a preferred embodiment of the invention.

Another modification to obtain selective replication in tumors is the elimination of adenoviral genes encoding related virus RNA (VA-RNA). These RNAS block the antiviral activity of interferon and, in the case of dellarovere, adenovirus becomes sensitive to inhibition by interferon. Because tumor cells are characterized by the restriction of the interferon pathways such adenoviruses replicate in tumors at a normal level. Thus, in another specific embodiment, selective replication in tumors is achieved by mutations in one or more genes of the group E1a, E1b, E4 and VA RNA of adenovirus. Preferred mutations in EA.

These two strategies to achieve selective replication in tumors are not mutually exclusive.

In another embodiment of the invention, the adenovirus comprises modifications in its capsid, increasing its infectious ability or the target is installed on its receptor, present in the tumor cells. In a preferred embodiment, adenoviral capsid proteins genetically modify to include ligands that increase the infective ability or that target the virus to a receptor in the tumor cell. Targeting of adenovirus to the tumor can be achieved by using bifunctional ligands that bind to the virus, on the one hand, and with the receptors of the tumor, on the other. On the other hand, to increase the stability of adenovirus in blood and, therefore, increase the possibility of reaching dispersed tumor inclusions capsid can be coated with polymers such as polyethylene glycol. In a preferred embodiment, the oncolytic adenovirus has the capsid modified to increase its infectious capacity or to improve its focus on target cells by replacing heparansulfate-binding domain KKTK in the fiber of adenovirus domain RGDK. In the examples illustrated construction of adenovirus ICOVIR17RGDK with such characteristics.

In another specific embodiment, the adenovirus contains a sequence that optimizes the transmission of a protein sequence that encodes a hyaluronidase.

In another specific embodiment, the adenovirus contains th is sequence, which activates the expression of sequences encoding hyaluronidase. In particular, this sequence selected from the group consisting of the sequence of splicing, which provides processing of RNA, the sequence of the IRES (internal plot ribosomal entry") and sequence 2A picornavirus.

In another specific embodiment, the oncolytic adenovirus contains other genes integrated in its genome, which are commonly used in the field of cancer gene therapy to increase the cytotoxicity of oncolytic adenoviruses in relation to the tumor cells. Some of them are gene timedancing, gene citizendumonde, genes proapoptotic, genes that stimulate the immune system, genes tumor suppressor genes or activating prodrugs.

These modifications in the genome of the adenovirus are not mutually exclusive. There are several ways to manipulate the genome of adenovirus. In the field of gene therapy and virotherapy using adenoviruses known methods of constructing genetically-modified adenoviruses. The most widely used method is based on constructing a first desired genetic modification in plasmids containing subject to modification region of the adenovirus, and then the implementation of homologous recombination in bacter the s using plasmids, containing the rest of the viral genome.

Adenovirus that contains a gene hyaluronidase is an object of the present invention, multiplies and amplificated cell lines commonly used in the field of gene therapy and virotherapy, such as cell line HEK-293 and A549. The preferred method of propagation is the method by infection of cell lines permissive for replication of the adenovirus. Cell line lung adenocarcinoma A549 is an example of a line with these characteristics. Reproduction is carried out, for example, as follows: A549 cells seeded in plastic tablets for cell culture plates and infect the rate of 100 viral particles per cell. Two days later, observed cytopathic effect, reflecting the production of the virus, in the form of clustering and rounding of cells. Cells are collected in test tubes. After centrifugation at 1000g for 5 minutes and the cell sediment is frozen and thawed three times to destroy the cells. The obtained cell extract was centrifuged at 1000g for 5 minutes, supernatant from virus layered on the gradient of cesium chloride and centrifuged for 1 hour at 35000g. The area of the virus obtained from the gradient layer to another gradient of cesium chloride and centrifuged again for 16 hours at 35000g. The area of virus is going to the t and deleteroute against PBS-10% glycerol. Cialisovernight virus divide into aliquots and store at -80°C. quantification of the number of viral particles and plaque-forming units is done according to standard protocols. Buffered phosphate saline (PBS) with glycerol to 5% is a standard structure for storing adenovirus. However, there have been described new compounds, which increase the stability of viruses. Methods purification of adenovirus containing the gene hyaluronidase, for its use in the treatment of cancer are the same as described for the other adenoviruses and adenoviral vectors used in virotherapy and gene therapy of cancer.

Oncolytic adenovirus of the present invention can be introduced mammal, preferably human. The purpose of introducing oncolytic adenovirus treatment, including, as non-limiting examples, melanoma, pancreatic cancer, colon cancer and lung cancer. It also covers the introduction of oncolytic adenovirus in tumors located in the precancerous stage. It is clear that oncolytic adenovirus is administered in pharmaceutically acceptable form. Specialists in this field can provide the required dose using standard procedures. It is clear that the dose should be an effective amount of oncolytic adenovirus to generate the, to ensure that the tumor in the patient's treatment. The virus can be injected directly into the tumor, into the cavity where the tumor is located in the vascular network of tumors around the tumor or by systemic intravenous injection into the patient. Preferably, the introduction of a system.

Protocols using viruses described in this invention, for the treatment of cancer are the same procedures that are used in the field of virotherapy using adenoviruses and gene therapy using adenovirus. In the field of gene therapy has a great experience of using noncritically and oncolytic adenoviruses. There are numerous publications describing the treatment of tumor cells in culture, animal models and clinical trials with patients. For treatment of cells in culturein vitroadenovirus purified using any of the compositions described above, is added to the culture medium to obtain infection of tumor cells. For the treatment of tumors in experimental animal models or in patients adenovirus can be entered by regional injection into the tumor or into the body cavity where the tumor or systemically by injection into the bloodstream.

Oncolytic adenovirus according to the invention can be entered separately or in whom oppozitsii with pharmaceutically acceptable carriers and excipients. Specialist in this field adjusts the composition in accordance with the specific way of introduction. The composition may include oncolytic adenovirus as a single anticancer agent or in combination with another therapeutic agent such as a chemotherapeutic drug or vector with integrated therapeutic gene. In addition, therapy with oncolytic adenoviruses can be combined with radiation therapy.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by any expert in this field. Methods and materials similar or equivalent to those described in the present description, can be used in the practice of the present invention. Throughout the description and claims the word "include" and its variations is not intended to exclude other technical features, additives, components or steps. Additional objects, advantages and features of the invention will become apparent to experts in this field after studying the description or may be learned from practice of the invention. The following specific embodiments of, and drawings are provided as illustrations, and they are not intended to be the trading to limit the present invention.

Description of the DRAWINGS

FIG.1 (a) shows the structure of oncolytic adenoviruses, characterized by content and gene expression of PH20 hyaluronidase. Adenovirus AdwtRGD-PH20 contains protein gene PH20, built after the gene of the adenovirus fiber. Expression of PH20 protein is regulated by the major late promoter (MLP) of adenovirus by inserting acceptor splicing (SA) of the adenovirus genome before PH20 protein. Protein translation of this gene is optimized by introducing a kozak sequence (k) before the sequence begins broadcasting. Adenovirus ICOVIR15 and ICOVIR17 are adenoviruses, can replicate selectively in tumor. They are characterized by a content of 4 E2F binding sites and one Sp1 binding site in the endogenous promoter EA. Both virus is also present a modified version of the viral fiber, in which was embedded the peptide RGD-4C, and the mutant variant protein EA who had deleterows amino acids 121-129 polypeptide chain (mutation Δ24). In addition, ICOVIR17 gene contains hyaluronidase PH20, built-in, as in the adenovirus AdwtRGD-PH20. (b) shows the sequence built into the adenovirus AdA24RGD instead of the sequence from nucleotide nucleotides 419 to 422. This insert is made to embed the four binding site to factor E2F-1 and one binding site to factor Sp1. Posledovatelno and, marked as "nt 385-419" and "nt 422-461" correspond to the wild type AdA24RGD. (c) shows the complete cassette integrated into the genome ICOVIR17 and AdwtRGD-PH20, relatively genomes ICOVIR15 and AdwtRGD (SEQ ID NO: 4). Shown acceptor splicing IIIa, kozak sequence and the polyadenylation (polyA). Sequence encoding a protein PH20, extends from kozak to a polyadenylation sequence. FIG.1 corresponds to EXAMPLE 3.

FIG.2 shows the amino acid sequence of the protein PH20 (SEQ ID NO: 1) and the hydrophobicity profile in accordance with the algorithm Kite Doolittle. Protein RN is a membrane protein present in plasma and acrosomal membrane of spermatozoa. (a) Amino acid sequence shows a hydrophobic sequence, responsible for zakalivanie protein in the membrane (underlined sequence). In the present invention expressed by the virus protein PH20 is deleteriously hydrophobic tail. The point of cleavage is indicated inside the circle. Due to this deletion PH20 protein is secreted in the extracellular environment. (b) hydrophobicity Profile 100 terminal amino acids of the protein PH20 Kite Doolittle. The arrow indicates the start of a hydrophobic portion which has been eliminated.

FIG.3 shows that oncolytic adenoviruses containing the gene hyaluronidase PH20, Express a soluble protein, the cat is who demonstrates the activity of hyaluronidase. The gels show that the samples of hyaluronic acid, incubated with the supernatant of the virus expressing hyaluronidase PH20, had split with the formation of oligosaccharides of various sizes. Samples incubated with supernatants control adenoviruses (AdwtRGD and ICOVIR15) demonstrate the presence of undigested hyaluronic acid. FIG.3 corresponds to EXAMPLE 4.

FIG.4 shows that the integration and expression of the gene hyaluronidase PH20 does not interfere with replication of adenovirus can replicate selectively in tumor. Cells cell lines A549 (a) and SKMel28 (b) infected oncolytic adenovirus ICOVIR15 and ICOVIR17 (which is different from ICOVIR15 content gene PH20) and the amount of virus in cell extracts was determined (the X-axis, a common virus, TU/ml) at different times (Y-axis in hours after infection). The graphs show that the kinetics of production of the virus is the same for both viruses, demonstrating that the incorporation and expression of hyaluronidase PH20 in the adenovirus ICOVIR17 does not affect viral replication. FIG.4 corresponds to EXAMPLE 5.

FIG.5 shows the oncolytic efficacy ofin vitrooncolytic adenovirus that contains and expressive gene hyaluronidase PH20. Oncolytic activity of adenovirus, espressimo hyaluronidase PH20 (ICOVIR17), compared toin vitrowith activity similar oncolytics the CSOs virus without gene hyaluronidase PH20 (ICOVIR15) in two tumor cell lines, espressiva large amounts of hyaluronic acid, SKMel28 (a) and PC3 (b). Induced by the virus cytopathic effect (CPE) was determined by lower levels of protein in the monolayer of infected cells was determined by BCA method). Cells were seeded into a 96-well plate in the amount of 10,000 cells/well. The next day the cells were infected serial dilutions of virus. Infected cells were incubated for 5 days, washed with PBS and determined the amount of protein remaining in the hole. The results show that in terms ofin vitroexpression of hyaluronidase PH20 does not improve oncolytic activity of adenovirus as cytotoxicity curves were similar for both viruses. % cell survival depending on TU/cell shown in the graph. FIG.5 corresponds to EXAMPLE 5.

FIG.6 shows the antitumor activity of oncolytic adenovirus, espressimo hyaluronidase PH20in vivo. Cells of human melanoma (SKMel28) was inoculated on each side of Nude Balb/c mice. When the tumors medium size 150 mm3in them were injected with PBS or 1×108transducers units AdwtRGD-PH20 (10 tumors/group). (a) the Graph shows the average tumor growth (in %) in each group relative to the day 0 as a function of time after injection (days). The result shows that the oncolytic adenovirus is, expressively gene hyaluronidase PH20, statistically has a higher antitumor activity compared with the control group (PBS), p<0,00001. 100% of tumors with injection AdwtRGD-PH20 regressed on 10-50% of the volume on the 27th day after injection, in contrast to 0% regression in the group with injection of PBS. (b) the amount of hyaluronic acid in tumors with injection of PBS or AdwtRGD-PH20 at the end of the experiment were analyzed immunohistochemically. The images demonstrate that tumors with injection AdwtRGD-PH20 have a lower amount of hyaluronic acid compared with the control tumors. FIG.6 corresponds to EXAMPLE 6.1.

FIG.7 shows that the expression of PH20 hyaluronidase enhances the antitumor effect of oncolytic adenovirus after intratumoral injection. Cells of human melanoma (SKMel28) was inoculated on each side of Nude Balb/c mice. When the tumors medium size 150 mm3in them were injected with PBS or 1×108transducers units ICOVIR15 or ICOVIR17 (10 tumors/group) in a single dose. (a) the graph shows the average tumor growth (in %) relative to day 0 as a function of time after injection (days). Oncolytic adenovirus, espessialy hyaluronidase PH20 (ICOVIR17), is a better antitumor effect than the control adenovirus that does not expressive this hialuronic the (ICOVIR15). (b) After 42 days of treatment, mice were killed and tumors were collected and weighed. The table shows the summarized results for the volume of the tumors, the percentage of tumor growth and weight of tumors at the end of the experiment. Tumors with injection ICOVIR17 are significantly lower tumor weight compared with tumors with injection ICOVIR15 (* p<0.05), and tumors with injection of PBS (# p<0,05). In contrast to the results obtainedin vitrowhere the virus can easily spread through the monolayer of cells, the results ofin vivoshow that inside the tumor, where the extracellular matrix prevents the spread of the virus, expression of PH20 hyaluronidase increases the antitumor activity of oncolytic adenovirus. FIG.7 corresponds to an EXAMPLE 6.2.

FIG.8 shows that the expression of PH20 hyaluronidase enhances the antitumor effect of oncolytic adenovirus after its systemic administration. Cells of human melanoma (SKMel28) was inoculated on each side of Nude Balb/c mice. After the tumors averaged 100 mm3in mice were injected with PBS or 5×1010physical particles ICOVIR15 or ICOVIR17 (ICOVIR15 containing PH20) (8-10 tumors/group) intravenously. (a) the graph shows the average tumor growth (in %) for each group compared with day 0 as a function of time after injection (days). The result shows that the expression of hyaluronan is duty PH20 leads to increased oncolytic activity of adenovirus, since the suppression of tumor growth induced ICOVIR17, significantly higher than the suppression induced in the control group (ICOVIR15), * p<0,00001. (b) Images show that the distribution of adenovirus ICOVIR15 and ICOVIR17 in tumors derived at the end of the experiment (day 48). Tumor mice with injection of oncolytic adenovirus ICOVIR17 demonstrate very extensive necrotic area (bold arrow), reducing the number of areas with viable cells (v) and large and numerous centers of virus replication (the area with the green fluorescence shows a thin arrows) compared with tumors with injection control adenovirus, ICOVIR15. FIG.8 corresponds to EXAMPLE 6.3.

FIG.9 shows that the increase in systemic antitumor activity of adenoviruses, espessialy the enzyme hyaluronidase PH20, not limited to one type of tumor. (a) the Graph shows the average growth of pancreatic tumors NP-18 (in %) for each group compared with day 0 as a function of time after injection (days). # indicates significant (p≤0,02) compared with tumors were treated with PBS from 14 to 30 day, and significant (p≤0,05) compared with tumors were treated with PBS from 14 to 30 days; *, significant (p≤0,02) compared with tumors that were treated with ICOVIR-15 from 12 to 30 day. (b) Images demonstrate distributed the e adenoviruses ICOVIR15 and ICOVIR17 in tumors NP-18 to 30. * p≤0,01 compared with tumors that were treated with ICOVIR15. "% p.a. the" mean % of positive area. FIG.9 corresponds to EXAMPLE 6.4.

FIG.10 (a) shows the structure of oncolytic adenovirus ICOVIR17 and ICOVIR17RGDK. (b) shows the amino acid sequence of a modified version of fibers in ICOVIR17RGDK. The underlined sequence corresponds to amino acids 91RGDK94 that differ from the fiber of the adenovirus type 5 wild-type person. FIG.10 corresponds to EXAMPLE 8.

FIG.11 shows the oncolytic activity of two adenoviruses (ICOVIR17 and ICOVIR17RGDK) in two tumor cell lines, one of which is a lung adenocarcinoma A549 (a) and the other pancreatic adenocarcinoma NP-18 (b). percent survival of cells depending on TU/cell. FIG.11 corresponds to EXAMPLE 9.

EXAMPLES

EXAMPLE 1. The design of oncolytic adenoviruses

Were designed two oncolytic adenovirus containing the gene hyaluronidase PH20: adenovirus AdwtRGD-PH20 and ICOVIR17.

cDNA hyaluronidase PH20 was obtained by NDP-amplification of different exons using as template genomic DNA of the cell line A549, and then connecting these specific exons flanking primers containing the restriction site Mfel. The resulting fragment was digested with Mfel and cloned the ay ligating in the Shuttle plasmid pNKFiberRGD (which contains the sequence of the fiber of adenovirus, modified RGD) to obtain plasmids pNKFiberPH20. cDNA corresponding PH20, cloned in plasmid pNKFiberPH20, represents in SEQ ID NO: 2. SEQ ID NO: 2 shows the encoding nucleotides for PH20 protein (isoform with inventory number GenBank NP_694859.1) from an initiation codon (ATG) to the position 1467. Nucleotide sequence region 1468-1527 this GenBank sequence encodes a hydrophobic end of the protein, which anchors the protein in the membrane. This sequence was demeterova, and it is not present in SEQ ID NO: 2. After nucleotide 1468 was added codon translation termination TAA.

EXAMPLE 2. Construction of adenovirus AdwtRGD-PH20: to create adenovirus AdwtRGD-PH20, fiber gene of adenovirus plasmids pVK50cau (which contains the full sequence of Ad5 with the restriction site Swa I in the fiber) was replaced by homologous recombination in the yeast genome fiber, followed by gene PH20 hyaluronidase derived from plasmids pNKFiberPH20, split NotI/kpni restriction sites.

Adenovirus AdwtRGD-PH20, characterized by expressionism gene hyaluronidase PH20 under the main control of the late promoter and content Tripeptide RGD in the fiber of adenovirus, was formed by cleavage using PacI plasmids pAdwtRGD-PH20 and transfection of HEK293 cells. Adenovirus AdwtRGD, described earlier, is characterized by the content of Tripeptide RGD in fiber adenov the Rus (see M. Majem et al., "Control of E1A to under an E2F-1 promoter to insulated with the myotonic 20 dystrophy locus insulator reduces the toxicity of oncolytic adenovirus Ad-Delta24RGD",Cancer Gene Therapy2006, vol. 13, pp. 696-705). AdwtRGD was constructed by splitting the Races I plasmids pVK503 containing the complete genome of the Ad5 fiber, modified RGD (see Dmitriev et al., "An adenovirus receiving-independent vector with genetically modified fibres 25 demonstrates expanded tropism via utilization of a coxsackievirus and adenovirus cell entry mechanism",J. Virol.1998, vol. 72, pp. 9706-13), followed by transfection of 293 cells.

EXAMPLE 3. Construction of adenovirus ICOVIR17: the formation of this adenovirus was used adenoviral plasmid pICOVIR17. To construct this plasmid gene of the adenovirus fiber of plasmids pICOVIR15 was replaced by homologous recombination in the yeast genome fiber with subsequent gene hyaluronidase PH20 from plasmids pAdwtRGD-PH20, split using SpeI/PacI.

Adenovirus ICOVIR15 derived from adenovirus AdA24RGD, which is characterized by the content deletions Δ24 in the coding sequence of the protein EA. This deletion affects the interaction EA with pRB. AdA24RGD also includes a box of RGD peptide in the fiber of adenovirus to increase infectious virus activity. These two modifications described in K. Suzuki et al., "Conditionally replicative adenovirus with enhanced infectivity 5 shows improved oncolytic potency",Clin Cancer Res2001, vol. 7, pp. 120-6. To control the expression of EA four of binding sites for E2F-1 and one part of the light is ivania Sp1 from AdA24RGD were integrated into the endogenous promoter EA. Thus was obtained ICOVIR15. This insert was made by replacing the sequence 419-422 genome sequence with 4 plots the binding of E2F-1 and one Sp1 binding site, so that is the final sequence, which is presented in SEQ ID NO: 3 and FIG.1(b). To implement this stage directed mutagenesis in the promoter EA plasmids pEndK/Spe was created unique BsiWI restriction site (see J. E. Carette et al., "Conditionally replicating adenoviruses expressing short hairpin RNAs silence the expression of a target gene in 15 cancer cells",Cancer Res2004, vol. 64, pp. 2663-7). The Sp1 binding site was introduced into the BsiWI restriction site of plasmid pEndK/Spe by ligating this split by BsiWI plasmids with primers Sp1F (5'-GTACGTCGACCACAAACCCC GCCCAGCGTCTTGTCATTGGCGTCGACGCT-3' SEQ ID NO: 5) and Sp1R (5'-GTACAGCGTCGACGCCAATGACAAGACGCTGGGCGGGGTTTGTGGTCGAC-3' SEQ ID NO: 6), hybridized to each other. Plots of the binding of E2F was introduced via the binding of primers E2FF2 (5'-GTACGTCGGCGGCTCGTGGCTCTTTCGCGGCAAAAAGGATTTGGCGCGTAAAAGTGGTTCGAA-3' SEQ ID NO: 7) and E2FR2 (5'-GTACTTCGAACCACTTTTACGCGCCAAATCCTTTTTGCCGCGAAAGAGCCACGAGCCGCCGAC-3' SEQ ID NO: 8), hybridized to each other to construct plasmids pEndK415Sp1E2F2. Next, the sequence CAU, which contains the elements necessary for replication of the plasmid in yeast (centromere, the area of Autonomous replication ARS and selective marker URA3) was introduced by homologous recombination in yeast to form plasmid pEndK415Sp1E2F2CAU. And finally, to construct pCOVIR15cau was carried out homologous recombination in yeast between plasmid pEndK415Sp1E2F2CAU, split Cloned, and adenoviral genome of adenovirus AdA24RGD. ICOVIR15 was obtained by transfection PacI-split pICOVIR15cau in HEK293 cells.

Virus ICOVIR17, which contains the same modifications that ICOVIR15 plus insert hyaluronidase gene after gene of the adenovirus fiber was formed by cleavage of the plasmid pICOVIR17 using PacI and transfection into cells NECK. The correctness of the genome AdwtRGD-PH20 and ICOVIR17 was checked by restriction analysis by Hind III. In addition, the region of the gene PH20 sequenced using specific primers.

The full cassette was integrated into the genomes ICOVIR17 and AdwtRGD-PH20 in comparison with genomes of ICOVIR15 and AdwtRGD shown in FIG.1 (c) and in SEQ ID NO: 4. Sequence encoding a protein PH20, is located between the kozak sequence and a polyadenylation sequence.

EXAMPLE 4. Expression of soluble protein with the activity of hyaluronidase using adenovirus that contains a gene hyaluronidase PH20

To demonstrate that adenovirus that contains a gene hyaluronidase PH20, expressive soluble protein with the activity of hyaluronidase, culture cell line A549 infected with viruses AdwtRGD, AdwtRGD-PH20, ICOVIR15 or ICOVIR17 using a multiplicity of infection, which provided more than 80% of infection (20 M. O. I). At 24 h after infection infected medium was replaced with fresh medium. Then, on proses is following another 24 h, fresh medium (or supernatant) was collected and concentrated by filtration on a column (Amicon Extreme (Millipore, Billerica, USA) according to manufacturer's instructions. Concentrated supernatant incubated overnight at 37°C with a solution of hyaluronic acid (1.5 mg/ml) in phosphate buffer (pH=6) containing 0.1 M NaCl and 0.05% BSA. Split hyaluronic acid were analyzed by electrophoresis in 15% wage polyacrylamide gel (see M. Ikegami-25 Kawai et al., "Microanalysis of hyaluronan oligosaccharides by polyacrylamide gel electrophoresis and its application to assay of hyaluronidase activity",Analytical biochemistry2002, vol. 311, pp. 157-65). Oligosaccharide cleavage products of hyaluronic acid digestion were fixed in the matrix of the gel in the solution alcian blue for 30 minutes Finally, the oligosaccharides were progressively silver nitrate. The result is shown in FIG.3. The results show that the supernatant of cells infected with adenovirus containing the gene hyaluronidase PH20 (AdwtRGD-PH20 and ICOVIR17), contains soluble protein, is able to cleave hyaluronic acid, a polysaccharide of high molecular weight) on oligosaccharides ranging in length from 5 to more than 50 units of disaccharide glycosides of repetitions.

EXAMPLE 5. No effect on viral replication and cytotoxicityin vitromediated oncolytic adenovirus, which espressive gene hyaluronidase PH20

To verify that the inclusion of gene hyaluronic acid is easy PH20 had no effect on viral replication, tumor cell lines A549 and SKMel-28 were infected with oncolytic adenovirus ICOVIR15 or ICOVIR17. Four hours after infection the infectious medium was replaced with fresh medium. Total cell extracts were collected at different time points after infection, and three times were freeze-thawed to release viruses. The number of viruses in cell extracts was determined by infection of HEK293 and anti-jacksonboro staining (see Majem M above). The result is shown in FIG.4. The introduction of gene hyaluronidase PH20 does not affect the replication of adenovirus ICOVIR17, because this virus belongs to the same replication that adenovirus control.

To demonstrate the validity of the expression of hyaluronidase PH20 on the cytotoxicity of oncolytic adenovirusin vitrocells from tumor cell lines PC3 and SKMel-28 were infected with serial dilutions of virus ICOVIR15 or ICOVIR17. Five and six days after infection, respectively, the amount of protein, as survival of the cells was evaluated using a spectrophotometer. The results are shown in FIG.5. Lytic activity ICOVIR17 in these two tumor lines is the same as the activity ICOVIR15, indicating that the expression of hyaluronidase PH20 does not have any oncolytic advantages in terms ofin vitro.

EXAMPLE 6. The use of packing services: carousels adenovirus, which contains a gene hyaluronidase PH20, for the effective treatment of tumors.

6.1. Experimentin vivowas performed using Nude mice breed Balb/c mice with transplanted tumors SKMel-28. In the amount of 5×106tumor cells cell line SKMel-28 was injected subcutaneously on each side of the mouse. After 21 days, mice with tumors (tumor volume 150 mm3) distributed in different experimental groups (n=10 per group). The tumor control group received a single intratumoral injection of buffered saline solution (20 µl). Mouse in the group being treated with AdwtRGD-PH20, received intratumoral injection (20 μl) 1×108transducers units of this virus to the tumor (equivalent to 2×109viral particles or century. o'clock). Tumors were measured every two or three days using calipers and tumor volume was calculated according to the formula: V (mm3)=A(mm)×In2(mm2)×p/6, where a is a more or longitudinal dimension, and represents the transverse size. FIG.6 shows the percentage of tumor growth compared to the beginning of treatment (day 0). The results are presented as mean ± standard deviation. The statistical significance of the differences between the results were calculated using the nonparametric test Mann-Whitney for mismatched data. Growth curves were compared with the use the of variance analysis. The results were considered significant if p<0,05. Treatment of tumors of the adenovirus AdwtRGD-PH20 led to regression of tumors in 100% of the treated tumors. % tumor growth was significantly less than in the control group, starting from the first days after injection. Analysis of the tumors at the end of the experiment showed a decrease in the amount of hyaluronic acid present in the extracellular matrix of tumors that were injected with AdwtRGD-PH20.

6.2. In another experiment, treatment was carried out by intratumoral injection ICOVIR15 or ICOVIR17. Tumor cell lines human melanoma SKMel-28 implanted in Nude mice Balb/C nu/nu and, once created, they were subjected to intratumoral treatment using PBS or 1×108transducers units viruses ICOVIR15 or ICOVIR17 (equivalent to 2×109viral particles or century. o'clock). The results are shown in FIG.7. Treatment ICOVIR17 showed oncolytic activity, which leads to inhibition of tumor growth, significantly different from control group (PBS), p<0,05. At the end of the experiment, tumors were removed and weighted. Table of FIG.7 shows the mean tumor volume, the percentage of tumor growth and weight of tumors at the end of the experiment. The weight of tumors by injection ICOVIR17 is much smaller than the mass of the tumors in the control group, PBS (# p<0.05) and ICOVIR15 (* p<0,05).

6.3. In another exp is Rimante treatment was carried out by systemic injection ICOVIR15 or ICOVIR17. Tumor cell lines human melanoma SKMel-28 implanted Nude mice Balb/C nu/nu and, once created, the animals were subjected to treatment by injection into the tail vein of PBS or 5×1010physical virus particles ICOVIR15 or ICOVIR17. The results are shown in FIG.8. Treatment ICOVIR17 demonstrated oncolytic activity, which led to the suppression of tumor growth that is significantly different from the control group, PBS (# p<0,0001) and ICOVIR15 (* p<0,00001). At the end of the experiment, tumors were removed and frozen for eight. Different areas of the tumors, frozen for eight, were treated with antibodies against hexane (capsid protein of adenovirus) and were stained with a contrast dye 4',6-diamidino-2-phenylindole. Antitumor activity ICOVIR17 correlates with replication of adenovirus on the intratumoral level, which was assessed in tumors obtained at day 48 after injection. Tumors were treated with ICOVIR17 demonstrate large necrotic areas, a better distribution of viruses and a large area of viable cells than tumors with injection ICOVIR15.

6.4. In another experiment, treatment was carried out by systemic injection ICOVIR15 or ICOVIR17 Nude nu/nu mice Balb/C mice implanted with tumor cell lines adenocarcinoma of the pancreas person NP-18. After tumors were established, designusing volume 60 mm 3animals were subjected to treatment through the tail vein using PBS or 5×1010physical virus particles ICOVIR15 or ICOVIR17 (10 tumors/group). The results are shown in FIG.9, which shows that the increase of antitumor activity of adenovirus, espressimo the enzyme hyaluronidase PH20, not limited to one type of tumor.

FIG.9(a) shows that the expression of hyaluronidase PH20 leads to increased oncolytic activity of adenovirus, compared with the PBS group and the group of viral control (ICOVIR15). # means significant (p<0,02) compared with tumors were treated with PBS from 14 days to 30. & indicates significant (p<0.05) as compared with tumors were treated with PBS from 14 days to 30. * means significant (p<0,02) compared with tumors that were treated with ICOVIR15 from the 12 th to 30-th day. On the 30th day the tumors were removed and frozen for eight, later treated with antibody against hexane and painted a contrasting dye, DAPI.

To quantify the level of intratumoral replication ICOVIR-17 five viable areas of each tumor were analyzed (7/10 animals per group) staining against hexane and the percentage of positive areas were determined by computerized image analysis (software ImageJ). The results of this analysis are presented in FIG.9(b), where Macheda, that tumor NP-18, the treated ICOVIR17, showed a significantly larger area of staining on the adenovirus compared with tumors treated ICOVIR15 (*, significant at p<0,01).

EXAMPLE 7. Toxicological profile of oncolytic adenoviruses, espessialy gene hyaluronidase

To ensure that the introduction of gene hyaluronidase does not significantly changes the nature of the toxicity induced by oncolytic adenovirus after intravenous injection, were used Syrian hamsters (Mesocricetus auratusbecause they represent an experimental model animal permissive for replication of human adenovirus. Hamsters are experimental animal model, permissive for replication of human adenovirus. Were used immunocompetent females aged 5 weeks (5-6 animals/group). They received a single dose of 4×1011C. H. ICOVIR15 or ICOVIR17 intravenously via the main vein at day 0 in 300 μl PBS. In the control group was administered the same volume of PBS. Five days after injection, animals were slaughtered and each received a total blood and serum using cardiac puncture for determination of parameters of hepatotoxicity (enzymes AST and ALT) and counting populations of blood cells by the method of flow cytometry (hemogram). At the same time perfectly and liver of animals and were fixed in 4% paraformaldehyde for staining hematoxylin/eosin.

The results of the study hepatotoxicity showed that in this model both the virus caused a certain degree of liver inflammation, with increased levels of transaminases AST and ALT. However, between the animals, which were treated with ICOVIR15 or ICOVIR17, differences were not observed. On the Hematology level of each of the viruses caused an increase in the population of neutrophils, basophils and monocytes, as well as the reduction in the number of platelets compared to control animals, but again, without any distinction between ICOVIR15 and ICOVIR17.

EXAMPLE 8. The design of the virus ICOVIR17RGDK

To create this adenovirus was used adenoviral plasmid pICOVIR17RGDK. In this plasmid the gene fibers of wild-type adenovirus 5 was replaced by a variant, modified by his heparansulfate-binding domain (amino acids 91KKTK94 in polypeptide sequence is replaced by 91RGDK94). Plasmid pICOVIR17RGDK was constructed by homologous recombination in yeast between the product partial cleavage pICOVIR17 by NdeI and split by the EcoRI plasmid pBSattKKT (containing the modified version of the fiber of adenovirus, as described in N. Bayo et al. "Replacement of adenovirus type 5 fibre shaft heparan sulphate proteoglycan-binding domain with RGD for improved tumour infectivity and targeting".Human Gene Therapy2009, vol. 20, pp 1214-21).

FIG.10 shows the position of the modification 91RGDK94 in the context ICOVIR17RGDK, as well as full sequence is lnost protein fibers in the adenovirus. Adenovirus ICOVIR17 contains the variant fiber gene of adenovirus, where the peptide RGD-4C (Cya-Asp-Cya-Arg-Gly-Asp-Cya-Phe-Cya; CDCRGDCFC, SEQ ID NO: 10) was built into the HI-loopknob-domain protein (hypervariable loop, evolutionary non-conservative and strongly exposed on the capsid of adenovirus). ICOVIR17RGDK quite similar ICOVIR17, except gene fibers, since only the fiber ICOVIR17RGDK differs from the adenovirus type 5 wild-type human replacement amino acid91KKTK94peptide91RGDK94with high binding affinity of integrin in shaft-domain protein (SEQ ID NO: 9).

EXAMPLE 9. Oncolytic efficacy of adenovirus with a modification of the capsid ICOVIR17RGDK

As shown in FIG.11, a modification of the capsid, which is present in ICOVIR17RGDK, does not change thein vitrocytotoxicity of oncolytic adenovirus containing and espressimo gene hyaluronidase PH20. Oncolytic activity of two adenoviruses that Express hyaluronidase PH20 (ICOVIR17 and 25 ICOVR17RGDK), were compared on two tumor cell lines, A549 derived from adenocarcinoma of the lung (FIG.11(a)) and NP-18, derived from adenocarcinomas of the pancreas (FIG.11(b)). The cytopathic effect induced by the virus, determined to reduce the amount of protein in the monolayer of infected cells (BCA method). Cells of the two tumor cell lines were seeded in 96-well plate from which asceta 10000 cells/well. The next day the cells were infected serial dilutions of virus. Infected cells were incubated for 6 days, washed with PBS and determined the amount of protein remaining in the hole. The results show that in terms ofin vitromodification of capsid does not significantly changes the oncolytic activity of adenoviruses.

EXAMPLE 10. Different Toxicological profile of oncolytic adenoviruses that Express the gene hyaluronidase

To assess the impact of modifications RGDK in the background oncolytic adenoviruses, espessialy hyaluronidase were used immunocompetent mice Balb/C mice without tumors. Used six males (7 animals/group). They received a single dose of 5×1010C. H. ICOVIR17 or ICOVIR17RGDK intravenously through the tail vein at day 0 in 150 μl PBS. On the 7th day (2 animals/group) and day 12 (5 animals/group) after injection the animals were killed and from each received total blood and serum using cardiac puncture for counting populations of blood cells by the method of flow cytometry (hemogram) and to measure the parameters of hepatotoxicity (enzymes AST and ALT). The results of this study showed that both virus have increased levels of enzymes on the 7th day. However, these levels had returned to normal values on day 12. Between groups ICOVIR17 and ICOVIR17RGDK n the observed significant differences, despite the fact that the trend of reducing the hepatotoxicity was observed in the group of animals with injection ICOVIR17RGDK, compared with the group ICOVIR17 (slightly lower levels of AST and ALT). As for the hematological profile of the animals on day 12 after injection, significant differences in white blood cells and platelets was not observed, except in the number of lymphocytes, which was lower in animals exposed to treatment ICOVIR17 than in animals of groups PBS and ICOVIR17RGDK.

1. Oncolytic adenovirus containing integrated in its genome sequence, encoding the enzyme hyaluronidase, for the treatment of cancer or precancer cancer, where expression of the enzyme is controlled by a promoter active in the cells of animals.

2. Oncolytic adenovirus under item 1, where the adenovirus is a human adenovirus.

3. Oncolytic adenovirus on p. 2, where the human adenovirus selected from the group consisting of serotypes of human adenovirus from 1 to 51 and hybrids of two or more different serotypes.

4. Oncolytic adenovirus on p. 3, where the adenovirus person belongs to serotype 5.

5. Oncolytic adenovirus under item 1, where the enzyme hyaluronidase is a testicular hyaluronidase mammal.

6. Oncolytic adenovirus under item 5, where the enzyme hyaluronidase is a testicular guy is laronidase person.

7. Oncolytic adenovirus under item 1, where the sequence encoding the enzyme hyaluronidase, contains a sequence with the elimination of the membrane-binding domain, which leads to the formation of soluble enzyme hyaluronidase.

8. Oncolytic adenovirus under item 1, where the sequence of the enzyme is embedded in oncolytic adenovirus after nucleotide sequence of the fiber of adenovirus.

9. Oncolytic adenovirus under item 1, where the promoter is selected from the group consisting of a cytomegalovirus promoter, the chief of the late promoter of adenovirus, SV40 promoter, promoter timedancing of herpes simplex virus, RSV promoter, promoter EF1-alpha promoter beta-actin promoter, IL-2 person, promoter of IL-4, IFN promoter, E2F promoter and promoter GM-CSF person.

10. Oncolytic adenovirus under item 1, where the adenovirus contains a tissue-specific or opukholespetsificheskaya promoter, where the promoter controls the expression of one or more group of genes E1a, E1b, E2 and E4 to achieve selective replication in tumors.

11. Oncolytic adenovirus on p. 10, where the promoter is selected from the group consisting of E2F promoter, telomerase hTERT promoter, tyrosinase promoter, promoter specific antigen prostate cancer, promoter of alpha-fetoprotein and promoter SOH-2.

12. Oncolytics the second adenovirus under item 1, where the adenovirus contains a mutation in one or more genes selected from the group E1a, E1b, E4 and VA RNA, to achieve selective replication in tumors.

13. Oncolytic adenovirus under item 1, where the adenovirus comprises modifications in the capsid to increase its infectious ability, or to target a receptor that is present in tumor cells, and where the modification of the capsid is a replacement for heparansulfate-binding domain CCTC present in the fiber of adenovirus, domain RGDK.

14. Oncolytic adenovirus on p. 13, where the modification of the capsid is a replacement for heparansulfate-binding domain KKTK present in the fiber of adenovirus, domain RGDK.

15. Oncolytic adenovirus under item 1, where the adenovirus contains a sequence that optimizes the transmission of a protein sequence that encodes hyaluronidase.

16. Oncolytic adenovirus under item 1, where the adenovirus contains a sequence that activates the expression of sequences encoding hyaluronidase.

17. Oncolytic adenovirus on p. 16, where the sequence that activates expression selected from the group consisting of the sequence of the acceptor splicing, which provides processing of RNA, the IRES sequence and the sequence 2A picornavirus.

18. Oncology is eticheski adenovirus under item 1, where the adenovirus contains one or more genes integrated in its genome selected from the group of genes proapoptotic, genes, stimulating the immune system, genes, tumor suppressor genes or activating prodrugs.

19. Pharmaceutical composition that includes a therapeutically effective amount of oncolytic adenovirus, as defined in any of paragraphs.1-18, together with pharmaceutically acceptable carriers or excipients, for the treatment of cancer or precancer cancer.

20. The use of oncolytic adenovirus, as defined in any of paragraphs.1-18, to obtain drugs for the treatment of cancer or precancer cancer in a mammal, including humans.



 

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7 dwg, 4 ex

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology. Described is molecule of chimeric nucleic acid of porcine circovirus (PCV2Gen-1Rep), which includes molecule of nucleic acid, coding porcine circovirus of type II (PCV2), which contains sequence of nucleic acid, coding protein Rep of porcine circovirus of type 1 (PCV1). Chimeric molecule of nucleic acid is constructed by replacement of gene Rep ORF1 PCV2 with gene Rep ORF1 PCV1. Invention also includes biologically functional plasmid or viral vector, which contain unique molecules of chimeric nucleic acids, suitable host cells, transformed by plasmid or vector, infectious chimeric porcine circoviruses, which produce suitable host cells, method of obtaining immunogenic polypeptide product with application of novel chimera, viral vaccines, protecting pig against viral infection or syndrome of postweaning multisystem wasting syndrome (PMWS), caused by PCV2, methods of protecting pigs against viral infection or postweaning multisystem wasting syndrome (PMWS), caused by PCV2, methods of obtaining unique chimera PCV2Gen-1Rep and the like. Invention can be applied in veterinary.

EFFECT: invention additionally includes novel method of increasing level of replication and PCV2 titre in cell culture.

21 cl, 2 dwg, 6 ex

FIELD: biotechnologies.

SUBSTANCE: pox virus of variolovaccine is proposed, which includes a defect F2L gene and a suicide gene. Pox virus has oncolytic activity. Besides, a reproduction method of such pox virus and its use for treatment of proliferative diseases or diseases with increased activity of osteoclasts is proposed.

EFFECT: improving compound application efficiency.

31 cl, 12 dwg, 3 tbl

FIELD: biotechnologies.

SUBSTANCE: created is recombinant pseudo adenoviral particle based on human being adenovirus genome of the 5-th serotype containing expressing cassette with haemagglutinin gene of influenza virus being included. As a haemagglutinin gene of influenza virus of B/Brisbane/60/2008 strain haemagglutinin gene with pre-optimised for expression in human being cells nucleotide sequence was used providing overexpression of haemagglutinin gene of influenza virus of B/Brisbane/60/2008 strain. Haemagglutinin gene of influenza virus of B/Brisbane/60/2008 strain with optimised nucleotide sequence was cloned in expressing cassette under promoter control and contains polyadenylation signal. Promoter is cytomegalovirus promoter, and polyadenylation signal is SV40. Expressing cassette is located in zone of E1 deletion of human being adenovirus genome of the 5-th serotype. Also method of use of recombinant pseudo adenoviral particle based on human being adenovirus genome of the 5-th serotype for induction of specific immunity to influenza virus B.

EFFECT: possibility of use in pharmaceutical industry for production of vaccine preparations.

6 cl, 9 dwg, 1 tbl, 4 ex

FIELD: biotechnologies.

SUBSTANCE: characterised is recombinant pseudo adenoviral particle based on human being adenovirus genome of the 5-th serotype and method of its use. Provided particle contains expressing cassette with haemagglutinin gene of influenza virus being included. As a haemagglutinin gene of influenza virus, haemagglutinin gene of A/Perth/16/2009(H3N2) strain with pre-optimised for expression in human being cells nucleotide sequence presented in SEQ ID NO:2. The specified haemagglutinin gene of influenza virus of A/Perth/16/2009(H3N2) strain is cloned in expressing cassette containing polyadenylation signal SV40 under control of cytomegalovirus promoter. Presented invention may be used for induction of specific immunity to influenza virus A of H3N2 subtype during injection in efficient quantity.

EFFECT: providing intense expression of recombinant haemagglutinin of the specified influenza virus.

6 cl, 9 dwg, 1 tbl, 4 ex

FIELD: biotechnologies.

SUBSTANCE: characterised is recombinant pseudo adenoviral particle based on human being adenovirus genome of the 5-th serotype and method of its use as a component for production of vaccine for influenza virus A of H1N1 subtype. Presented recombinant particle contains expressing cassette including SV40 polyadenylation signal and cytomegalovirus promoter with influenza virus haemagglutinin gene being included. As influenza virus haemagglutinin gene, haemagglutinin gene of strain A/California/07/2009(H1N1) is used with pre-optimised for expression in human being cells nucleotide sequence provided in SEQ NO:2. These inventions allow raising specific immunity to influenza virus A of H1N1 subtype by provision of overexpression of haemagglutinin gene of influenza virus A/California/07/2009(H1N1).

EFFECT: improvement of the method.

6 cl, 9 dwg, 1 tbl, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and concerns cDNA coding human dysferlin, a genetically engineered construct wherein such cDNA is cloned, a recombinant adenovirus and a pharmaceutical composition. The described genetically engineered construct comprises an expression plasmid adenovirus vector pAd/CMV/V5-DEST, wherein the codon-optimised cDNA having a sequence presented in SEQ ID NO: 1 and coding human dysferlin is cloned according to recombination sites attB1 and attB2. The recombinant replication defect adenovirus serotype 5 is prepared with using such genetically engineered construct and included into the pharmaceutical composition in an effective amount.

EFFECT: inventions enables recovering the disturbed dysferlin expression and/or function in the skeletal striated muscle tissue and resulting in the positive effect.

4 cl, 2 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and represents an oncolytic adenoviral vector, a cell and a pharmaceutical composition containing the above vector, as well as applications of the above vectors in preparing a drug for treating cancer in an individual, and to a method of treating cancer in the individual. The presented invention may be used for treating cancer. The oncolytic adenoviral vector contains a nucleic acid skeleton of serotype 5 adenovirus (Ad5), deletion in 24 nucleotides related to amino acids 122-129 in E1A constant region 2, and a nucleic acid sequence coding the human granulocyte-macrophage colony-stimulating factor (GM-CSF) in a site of deleted gp19k/6.7K in the region E3, and capside modification if needed.

EFFECT: invention provides improving the tumour-specific immune response in the individual.

40 cl, 36 dwg, 10 tbl, 7 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology. What is presented is an adenovirus helper vector for preparing a high-capacity recombinant adenovirus. The invention can be used in cell technology.

EFFECT: said vectors enable developing systems for creating high-capacity recombinant adenoviruses in the cells, which optimally decrease contamination of the above high-capacity adenoviruses by the helper adenoviruses.

28 cl, 14 dwg, 5 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to genetic engineering and can be used to optimise expression of the antigen protein of the human epidermal growth factor-2 (HER2/neu). To obtain the HER2/neu protein, a nucleic acid synthetic molecule is used, which is codon-optimised for high level of expression of the said protein in a human cell.

EFFECT: invention increases production of the recombinant HER2/neu protein during expression in human cells.

8 cl, 10 dwg, 14 ex

FIELD: gene engineering, in particular apoptosis inducing gene delivery vectors useful for cancer, hyperplasia, metaplasia and displasia diagnosis and treatment.

SUBSTANCE: recombinant adenovirus apoptin-containing vectors are obtained by cotransfection into 911 helper cell line of p.Amb-VP3 adaptor plasmids (in case of VP3 protein expression) or pMAb-VP2 plasmids (in case of VP2 protein expression) and JM17 DNA. p.Amb-VP3 plasmids carry apoptin gene in 5'-3'-orientation, expressing under control of adenoviral main late promoter. Plasmid JM17 DNA contains complete adenoviral DNA excepted E1 and E2 regions. pMAb-°VP2 plasmids carry apoptin gene with two point mutation in limits of coding region. Cotransfections are carried out by calcium phosphate method. Recombinant adenoviral DNA is formed by homologous recombination between homologous viral sequences representing in p.Amb-VP3 (or pMAb-VP2) plasmid and in adenoviral DNA from plasmid JM17 DNA. Cell infection of various human tumors with gene delivery vectors causes to tumor cell apoptosis induction and sufficiently reduced normal, diploid, non-transformed or non-pernicious cell apoptosis.

EFFECT: new gene delivery vector capable to induce cell apoptosis.

8cl, 7 dwg

The invention relates to new viral vectors

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

FIELD: chemistry.

SUBSTANCE: invention relates to a compound of formula wherein each of R1 and R2 is independently selected from a group consisting of a hydrogen atom, nitro and NR6R7; R3 is C1-C8alkyl; each of R4 and R5 is independently selected from a group consisting of C1-C8alkoxy, phenoxy and phenyl(C1-C8alkylene)oxy; each of R6 and R7 is independently selected from a group consisting of a hydrogen atom, C1-C8alkyl, C(O)R8 and SO2R8;R8 is selected from a group consisting of a hydrogen atom, C1-C8alkyl, halogen-substituted C1-C8-alkyl, C1-C8-alkyl, substituted (C1-C8-alkylsubstituted amino), C1-C8-alkyl, substituted with piperidine and C1-C8-alkyl, substituted with morpholine.

EFFECT: reduced PDE4 enzyme activity and treating PDE4 enzyme mediated diseases or conditions.

21 cl, 2 tbl, 32 ex

FIELD: chemistry.

SUBSTANCE: invention relates to application of 5-amino-3-(2-aminopropyl)-[1,2,4]thiadiazole derivatives of general formula (I) as cytostatic preparations for fighting oncologic process in form of bases or pharmacologically acceptable salts. In formula (I) R1, R2 can be similar or different and independently represent hydrogen, halogen, alkyl, R3 represents alkyl, aralkyl, heteroalkyl, cycloalkyl.

EFFECT: increased efficiency of composition application.

2 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to compounds of structural formula

possessing inhibitory activity on BTK, TEC, BMX, ITK, ErbB1, ErbB4 and/or JAK3 kinases. In formula (I-b), ring A and ring B represents phenyl; Ry represents -CN, -CF3, C1-4 aliphatic group, C1-4 halogenaliphatic group, -OR, -C(O)R or -C(O)N(R)2; each group R independently represents hydrogen or a group specified in C1-6 aliphatic group optionally containing a substitute presented by halogen, -(CH2)0-4R°, -(CH2)0-4OR°, -(CH2)0-4N(R°)2, -(CH2)0-4N(R°)C(O)OR°, -(CH2)0-4C(O)R°, -(CH2)0-4S(O)2R°, or 5-6-merous substituted or aryl ring containing 1-2 heteroatoms independently specified in nitrogen or oxygen optionally substituted by group =O, -(CH2)0-4R°, -(CH2)0-4N(R°)2 or -(CH2)0-4OR°; phenyl; 5-6-merous heterocyclic ring containing 1-2 heteroatoms independently specified in nitrogen, oxygen or sulphur optionally substituted by group -(CH2)0-4R°, -(CH2)0-4OR° or =O; or 6-merous monocyclic heteroaryl ring containing 1 nitrogen atom; W1 and W2 represent -NR2-; R2 represents hydrogen, C1-6aliphatic group or -C(O)R; m and p are independently equal to 0, 1, 2, 3 or 4; Rx is independently specified in -R, -OR, -O(CH2)qOR or halogen, wherein q=2; Rv is independently specified in -R or halogen; R1 and R° radical values are presented in the patent claim. The invention also refers to a pharmaceutical composition containing the above compounds.

EFFECT: preparing the compounds possessing the inhibitory activity on BTK, TEC, BMX, ITK, ErbB1, ErbB4 and/or JAK3 kinases.

17 cl, 25 dwg, 20 tbl, 286 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a set, containing calcium sulphate hemihydrates, pressed particles of calcium sulphate dehydrate, additionally containing one or more therapeutically, preventively and/or diagnostically active substances, and sodium-carboxymethylcellulose (Na-CMC) and a water medium, including water. The ratio R of sodium-carboxymethylcellulose and calcium sulphate in the set constitutes from 0.1 mg of sodium-carboxymethylcellulose (calculated as Na-CMC)/g of calcium sulphate to 8 mg of sodium-carboxymethylcellulose (calculated as Na-CMC)/g of calcium sulphate. When mixed, the said components of the set form a bioresorbable ceramic composition. The invention also relates to the application of the set for the treatment of a disease or condition, associated with prostate. Also claimed is a composition ready for application in the form of a paste for introduction to a patient during the time period from 5 minutes to 1 hour before hardening, obtained by mixing the components of the set. Also claimed are: a hardened composition and a method of obtaining the hardened composition or the composition ready for application.

EFFECT: control of the time of the set and composition hardening.

13 cl, 12 tbl, 9 ex

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