Method of obtaining system of directed delivery of protein molecules (oncolytic proteins) into tumour cells based on activated lymphocytes

FIELD: medicine.

SUBSTANCE: invention relates to the field of biotechnology, in particular to the lentiviral delivery of apoptin into tumour cells, and can be used in medicine. The method includes obtaining a lentiviral construct, expressing modified apoptin, fused with a sectretory signal of lactotransferrin and a transduction signal (ST-CTP-apoptin), with the further obtaining of recombinant lentiviral particles, defective by replication and carrying the modified apoptin, which are later introduced into T-lymphocytes (TILs), obtained in the surgical ablation of the tumour or in the process of obtaining a biopsy, possessing the ability to penetrate into tumour cells. After that, obtained TILs are autotransplanted to the said patient.

EFFECT: invention makes it possible to increase the ability of apoptin to penetrate into tumour cells and produce an oncolytic effect with respect to all the tumour cells.

2 dwg, 3 ex

 

The invention relates to the field of medicine and biotechnology.

Modern methods of cancer therapy based on the application of an integrated approach using a combination of funds focused on certain features of the biological properties of tumor cells. With the deepening of knowledge about tumor cells and determine the most universal mechanisms that distinguish cancer cells from normal, it becomes possible to develop more effective, specific and safe for the body biological therapy. In contrast to the lack of specific existing chemotherapeutic agents, biological therapies are designed to use natural activity and mechanisms to identify and kill cancer cells.

Currently more and more attention of researchers is attracted by the prospects of using approaches to therapy of malignant tumors, based on the use of biomolecules - proteins with selective inhibitory or even toxic effects against tumor cells, while not exerting similar effects on normal cells. Such approaches include the use of specially designed antibodies against ofwholesale epitopes that inhibit tumor growth or by blocking the re�of atarov, necessary for active proliferation of the tumor, either by direct cytotoxic effect of the toxin is covalently linked to the antibody. Another group of protein molecules, with no less potential use in therapy, oncolytic are protein molecules which when injected into the tumor cell, initiating a cascade of Pro-apoptotic signals, dependent on pathological signaling pathways characteristic of transformed cells, but not normal.

The main advantage of protein molecules compared to the traditionally used cytotoxic chemical compounds is that the mechanism of their action is aimed at fundamental features of cancer cells, which provides exceptionally high selectivity of their action, in combination with low toxicity for the body and no side effects. Due to this, a strong cumulative effect of several drugs on the tumor can be achieved without complications, usually observed in cancer therapy.

In recent years more and more works describe the inactivation of tumor suppressor p53 as one of the main causes of cancer. As you know, p53 is a key component responsible for launching of protective signaling pathways in response to DNA damage in mammalian cells. �adaplene damage in the genetic apparatus of the cell leads to activation of p53 protein, resulting in a stopping of the cell cycle and DNA replication, with strong damage occurs programmed cell death or apoptosis. Thus the mechanism that can prevent and remove potentially oncogenic cells, and mutation or loss of p53 gene leads to the accumulation of a multitude of DNA damage, cell degeneration and, eventually, to cancer. About 50% of all malignant tumors detected mutation namely p53 gene. Therefore, of great scientific and applied importance are studies devoted to the search for alternative, p53-independent pathways of apoptosis. Recently it was found that such a mechanism has apoptosis induced by Apoptin.

Apoptin (product VP3 gene of the genome of the virus of infectious anemia CSD) - Proline-rich protein with a molecular mass of 13.6 kDa, consisting of 121 amino acid residues.

Greater interest is the ability apoptin to selectively induce apoptosis of transformed cells of birds and mammals, while not affecting normal, untransformed cells. Based on the obtained data, we can speak about 70 different tumor lines that are sensitive to apoptin. Apoptin region has nuclear localization, which is located in the C-terminal region of the protein between 80-121 amino acid residues, and the area of nuclear�exports, located in the Ν-terminal region, between 33 and 46 amino acid residues. Many studies have shown that nuclear localization apoptin in tumor cells is required for induction of apoptosis, whereas in normal cells the protein is localized mainly in the cytoplasm, where it is unstable and rapidly degraded.

As already mentioned, VP3 can cause the death of cancer cells while not affecting normal. Such selectivity may explain the Nucleo-cytoplasmic transport. Experimental data showed that nuclear export apoptin in normal cells is identified by a unique signal sequence (nuclear export sequence) interacts with exportin CRM1, exercising, thus, the permanent withdrawal of protein from the nucleus. In cancer cells the reverse transfer does not occur, due to phosphorylation 108 threonine (Thr 108) in the nuclear export sequence, which blocks the interaction with CRM1. Thus, feofilivka Thr-108, as it seems, may be one of the key events required for the accumulation apoptin in the nucleus and apoptin-induced apoptosis. Further, more detailed study of this phenomenon showed that the phosphorylation apoptin is not a prerequisite of its tumor-specific on�of aplane in the kernel. Thus, replacement 107 and 108 of threonine for alanine residues, and further validation of the subcellular localization of the mutant apoptin in MCF-7 cells and normal cells did not reveal any changes. This protein still accumulated in the nuclei of tumor cells and subsequently lead to apoptosis. This indicates the existence of other mechanisms of accumulation apoptin in the nuclei of tumor cells.

Medical application of oncolytic proteins, including apoptin, while complicated by the low efficiency of the used methods of delivering biological agents in the areas of the tumor. Another challenge is to deliver the protein directly to tumor cells, because unlike small chemical molecules, proteins lack the ability to penetrate through the plasma membrane. A significant limitation is the immunogenicity of proteins and the rapid formation of antibodies, which, over time, can completely neutralize the activity of oncolytic protein. In order oncolytic proteins become effective anticancer therapy, we need to find ways to effectively address these problems: to determine modifications of protein structure, necessary for its smooth passage through the plasma membrane and to develop a fundamentally new means of targeted delivery of such therapeutic use�quarter of agents to tumor cells. These delivery methods have, on the one hand, to protect the protein from the action of antibodies, and to bring the release of the active protein to the location of tumor cells.

Currently there are attempts to adapt apoptin as the most promising oncolytic protein for clinical use.

Having a high selectivity against tumor cells, apoptin has good prospects for application in cancer therapy. To ensure delivery apoptin in tumor cells, you must either enter into these cells a genetic construct expressing apoptin, or to modify the structure apoptin, giving it the ability to penetrate into tumor cells through the plasma membrane. The first method of delivery used in cancer therapy using oncolytic viruses, while the second method of delivery allows you to use apoptin a variety of ways: traditionally, introducing synthetic recombinant apoptin, and using biological means of delivery.

Medical application prospects apoptin and oncolytic proteins, in General, depend upon the development of the means of delivery of recombinant proteins in the tumor. Injecting the synthesized protein is not n�will create a local high concentration in the tumor and bears the risk of immunization the patient to input a squirrel or development of allergic reactions. To administer an injection directly into the tumor is not always possible, in addition, this injection may cause a number of complications. The best option the use of oncolytic proteins would be the presence in the tumor cells producing these proteins. In this case, the impact would be long, and the concentration of the drug is maximal in the region of the tumor. To achieve this purpose, commonly used recombinant viral vectors are collected on the basis of lentivirus or adenovirus.

Vectors based lentivirus described, for example, Ye Feng, etc., Construction of three recombinant lentiviruses containing apoptin driven by survivin promoter of different lengths and comparison of their tumor suppressing efficency in vitro, Journal of Third Military Medical University, 2012-01. Lentiviral vectors will allow you to write and to Express the constitutive recombinant apoptin in tumor cells and surrounding tissues. This expression will be longer, hazards of immunization against the virus almost never occurs because he is not able to replicate. However, the nature of integration of viral DNA into the genome carries the risk of damage and, as a consequence, additional malignancy of the tumor.

Known patent RU 2252255 describing recombinant adenoviral vector containing apoptin for the treatment of cancer, which are obtained by cotransfection in a helper cell line 911 adaptor plasmids�p ID.AMb-VP3 (in the case of expression of protein VP3) or plasmids pMAb-VP2 (in the case of VP2 protein expression and plasmid DNA JM17. Plasmids pAMb-VP3 gene are apoptin in orientation 5'-3', the expression of which is under the control of adenovirus late chief promoter. Plasmid DNA JM17 contains the complete DNA of adenovirus with the exception of region E1 and E3. Plasmids pMAb-VP2 gene are apoptin, with 2 point mutations within the coding region.

Vector delivery of genes capable of inducing apoptosis in a cell comprising the DNA fragment encoding the protein having adapteroptions activity, and apoptosis is induced in cancer cells and to a lesser extent or not at all in normal untransformed diploid/non-cancerous cells, wherein the gene delivery vector is independent of the infective vector and represents the adenovirus.

A significant disadvantage of this method is gradually emerging immunization of patient-specific adenoviral strain.

Known patent RU 2492238 C1, which relates to recombinant plasmid DNA pGEM-Puro-DS-Apo, carrying the synthetic gene apoptin size 363 BP with a specific nucleotide sequence, flanked by fragments of the genome of vaccinia virus has a size 6298 D., a molecular weight of 4.19 MDA, and recombinant strains of vaccinia virus producing apoptin anemia virus of chickens. Using this method will be n�to blogatize also gradually emerging immunization of the patient.

Described drawbacks necessitate the development of a new, non-viral, method of delivery and expression of recombinant proteins in tumors

Known patent US 5656465 concerning ways to deliver genes in vivo. Describes the use of non-cumulative viral vectors having a low replicative efficiency for inserting a gene into a cell such as a lymphocyte or a tumor cell is a privileged system for transforming such cells for use in somatic cell therapy or gene therapy. Genes are inserted into the target cell, in particular in the target cell, representing the lymphocytes infiltrating the tumor tissue. It is noted that the interest are genes encoding TNF, TGF-α, TGF-β, hemoglobin, interleukin-1 and the like, as well as biologically active mutiny of these proteins. Not mentioned the possibility of targeted delivery apoptin.

The object of the present invention is to provide a new means of targeted delivery apoptin to tumor cells, which allows, on the one hand, to protect the protein from the action of antibodies, and to bring the release of the active protein to the location of tumor cells.

Technical result: increased ability apoptin to penetrate into tumor cells and provide oncolytic effect in respect of� all of the tumor cells. This therapy can be used in combination with other anticancer drugs that will reduce the duration of treatment and reduce side effects.

The problem is solved by a method for producing the targeted delivery system apoptin in tumor tissue. Method harakteryzuyetsya that modify apoptin by fusion of secretory signal lactotransferrin and transductional signal (ST-CTP-apoptin), get a lentiviral construct expressing ST-CTP-apoptin specified construct is used to produce recombinant lentiviral particles defective in replication and load-bearing modified apoptin, which are then injected into autotransplantation lymphocytes infiltrating in the tumor tissue.

As a means of delivery and the producer of oncolytic protein proposed autotransplantation lymphocytes infiltrating in the tumor tissue (TIL, tumor infiltrating lymphocytes). Lymphocytes can be obtained during surgical removal of the tumor or in the process of obtaining the biopsy. Can also be used isolated from peripheral blood lymphocytes, activated by cultivation in the presence of interleukin-2. Such cultivation enhances the tropism of lymphocytes to the tumor, and, after entering back into the bloodstream, a significant portion of lymphocytes localized in tumor tissue.

<> The main difference of the present invention is the use onkologicheskih lymphocytes as a carrier and the producer apoptin - is new and not described in the literature. This approach will allow to combine oncolytic effect apoptin with the antitumor effect of cell and immunotherapy of malignant tumors. Unlike the known solutions in that it describes the constructs reintegriruet into the genome of the vector, while the stated lentiviral construct is integrated.

To solve the problem were developed signal peptide ensuring secretion of the expressed oncolytic protein from cells and subsequent penetration into the surrounding cells by protein domain that provides penetration through the plasma membrane. In the invention used property of cytotoxic lymphocytes to infiltrate and accumulate in the tumor. Expression of secreted oncolytic lymphocyte protein would release active therapeutic molecules directly at the location of the cancer cells and reduce the probability of neutralization of the active protein antibodies.

The invention is demonstrated below by examples.

Example 1. Determination of the optimal variant of recombinant apoptin have�its maximum oncolytic activity

In cDNA apoptin PCR integrated domain cytoplasmic transduction (P) and secretory signal, and the resulting sequence is cloned in the lentiviral vector. Created a series of constructs containing different variants of the P-domain (option 4, a fragment of the TAT protein of HIV (PTD domain) and 3 synthetic peptide), and variants of the ST-peptide (5 options, 3 of which are synthetic, derived on the basis of the predictions of the hmm-algorithm for determining the effectiveness transducing peptide, and 2 options are taken from natural secreted proteins, cystatin C and lactotransferrin). Series obtained constructs introduced into cells-producers, and one of them made the selection of the most effective option. This is filtered by the environment in which producers were cultivated, processed cell cancer cell lines SAOS2 and RKO, and comparative evaluation of cytotoxicity of each variant protein. This approach allows us to determine the functional activity of the protein in conditions similar to real. As control structures, as well as visual evaluation of the efficiency of secretion and capture of the modified protein, the resulting set of strains, secreting fluorescent protein, fused with a test PAGE - and ST-domains.

Has created a series of genetically engineered structures, designed to Express�Yu different options oncolytic protein apoptin and red fluorescent protein tagRFP. Were created constructs expressing wild apoptin (wt-apoptin), apoptin, merged with secretion signals (ST-apoptin), obtained from proteins lactoferrin, cystatin C, as well as three synthetic ST-signals obtained on the basis of the predictions of the hmm-algorithm for determining the efficiency of the secretory peptide. Also the resulting construct expressing apoptin, merged with a strong cytoplasmic transduction is one option, which is a fragment of TAT protein of HIV (PTD domain), and three synthetic peptide. In addition to constructs expressing apoptin fused separately with signals secretion and cytoplasmic transduction (5 and 4 items, respectively), were obtained expressone vectors containing all versions apoptin, and merged with secretory and transductional signal at the same time.

All apoptin-expressing constructs were introduced by the method of lentiviral transfection in culture tumor cell lines SAOS2 and RKO, and comparative evaluation of cytotoxicity of each variant protein. To do this, the obtained recombinant lentiviruses, after concentration with polyethylene glycol was determined the titer (by RT-PCR in real time), and then, after infection of cell cultures with an equal number of viral particles was determined, in which case the cytotoxicity Mac�Kalina. In fact, various options apoptin showed a cytotoxicity of less than "wild" apoptin (which was expected), but some options, including apoptin fused with the secretory signal lactotransferrin and transductional signal PTD showed an almost unchanged level of cytotoxicity.

In parallel with these experiments, cell lines SAOS2 and RKO were transfected by tagRFP-containing structures. After the selection and sorting, in which 90-95% of the cells in the culture became tagRFP-expressing cells were mixed in a ratio of 1/1 with nitrostilbene SAOS2 and RKO, respectively. After co-culturing for 24 hours, comparative fluorescence of two populations of co-cultured cells was changed on the cytometer. On the basis of the gain characteristic tagRFP fluorescence in nitrostilbene the clack, conclusions were drawn about the effectiveness of a couple-secretory and transducing peptides. The experiments showed that the greatest efficiency of the tested variants has a couple of secretory signal lactotransferrin and transductores PTD signal.

The sequence apoptin with secretory and transductive peptides SEQ ID NO 1 shown in Fig. 1. Example expressing constructs containing apoptin with a signal peptide, �of redstapler in Fig. 2.

Thus, it was determined the most effective structure through which onkologicheskie proteins can be introduced into tumor cells - ST-CTP-apoptin. The expression of ST-CTP-apoptin in a small subset of tumor cells, or cells of the tissue adjacent to it, leading to long-term presence of high concentrations apoptin in the intercellular space of the tumor.

Example 2. The cultivation and activation of lymphocytes in model animals, lines Nude thymus of mice

The homozygous Nude thymus of mice was inoculated with the tumor, in parallel, in heterozygous animals of the same line from peripheral blood were isolated mononuclear cell fraction. Lymphocytes were transducible the lentiviral expression tagRFP, and subjected to cultivation with different activating conditions (phytohemagglutinin, IL-2, IL-4, GM-CSF, lectins). After the operating time of a sufficient number of activated lymphocytes, they were introduced to Nude thymus mice with grafted tumors. Then, the removed tumor was analyzed on a flow cytometer, for the purpose of determining the amount of the contained fluorescent lymphocytes.

Example 3. A comparative study of the antitumor efficacy

Lentiviral construct expressing ST-CTP-apoptin, was introduced in lymphocytes in parallel with the control lentiviral construct. TC�tki were subjected to identical conditions of cultivation and inoculated into animals with tumors. Antitumor efficacy of ST-CTP-apoptin assessed in vivo by measurement of the dynamics of tumor development in mice that received apoptin-expressing and control lymphocytes expressing fluorescent protein. Introduction lymphocytes expressing apoptin leads to significantly higher efficiency of this method against a panel of human tumor cells, cultured on Nude thymus mice.

A method of obtaining a system of targeted delivery apoptin in tumor tissue, comprising a lentiviral construct expressing a modified apoptin, characterized in that the modified apoptin is apoptin fused with the secretory signal lactotransferrin and transductional signal (ST-CTP-apoptin) and having the sequence SEQ ID NO. 1 shown in Fig. 1, get a lentiviral construct expressing ST-CTP-apoptin with the arrangement of the elements shown on Fig. 2, followed by production of recombinant lentiviral particles defective in replication and load-bearing modified apoptin, which are then injected into T-lymphocytes (TILs) obtained during surgical removal of the tumor or in the process of obtaining a biopsy, which have the ability to penetrate into tumor tissue, with subsequent autotransplantation of these lymphocytes to ocosandomeprazole.



 

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33 cl, 3 dwg, 1 tbl, 2 ex

FIELD: biotechnology.

SUBSTANCE: invention represents a culture medium for the micropropagation of calciphilic plants in culture in vitro, comprising vitamins and amino acids dissolved in distilled water, on Murashige and Skoog, sucrose in an amount of 20,000 mg/l, agar-agar in an amount of 8,000 mg/l, and also growth regulators 6-benzylaminopurine, gibberellic acid, and 3-indolyl acetic acid, the mineral composition Woody Plant Medium, kinetin, at that the growth regulators are taken in the following concentration, mg/l: 6-benzylaminopurine 0.1-0.3, kinetin 0.9-1.1, gibberellic acid 0.9-1.1, 3-indolyl acetic acid 0.4-0.6.

EFFECT: invention enables to increase the propagation factor by activating the axillary meristems without compromising on the quality of regenerants.

2 tbl

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