RU2697797C2 - Genetic construct for inducing proliferation of peripheral monocytes in vitro - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: disclosed is a genetic construct for inducing the proliferation of peripheral monocytes and dendrite cells in vitro derived from human blood, comprising sequences coding transcription factors c-Mus and BMI1, as well as their binding self-splitting peptide sequence of Thosea asigna T2A virus, providing polycistronic expression of transcription factors under the control of one EF1alpha promoter. Said construct is derived from expression plasmid lentiviral vector pLEF1a-tagRFP, including a number of regulatory sequences: 5' and 3' LTR long end repeats, Rev dependent element (RRE or Rev-response element), Env is a regulatory sequence providing packaging of a viral genome into a viral particle structure with formation of a virion, cPRT - central polypurine path, a post-transcriptional regulatory element WPRE, an EF1alpha promoter.EFFECT: invention provides effective constitutive expression of transcription factors s-Mus and BMI1 when introducing said construct into peripheral monocytes and dendritic cells.1 cl, 3 dwg, 3 ex

Description

The invention relates to the field of biotechnology and can be used to increase the efficiency of the preparation of dendritic cell and T-cell vaccines for the treatment of malignant neoplasms.

Success in studying the nature of malignant tumors, the identification of molecular and genetic signs of a tumor predetermine the search for fundamentally new methods of treatment, which are based on selective damage to cells that are genetically different from normal body cells. One of these methods is immunotherapy, which is based on the activation and intensification of the processes of a specific immune response of the body to tumor development.

This method consists in the activation of ex vivo tumor antigens of elements of the immune system with the production of tumor-specific antibodies or effector cells, followed by their introduction into the patient's body.

Today, there are a large number of antibody preparations, some of which have already successfully passed clinical trials. However, no significant increase in patient survival was found.

Another potentially effective area of immunotherapy is the use of dendritic cells (DC). Dendritic cells play a key role in the regulation of acquired immunity, providing effective protection of the body against many infections and various pathologies. The main function of dendritic cells is to absorb foreign antigens, their processing and presentation of antigenic determinants to the effector cells of the immune system. In the context of oncological diseases, dendritic cells are extremely promising therapeutic agents, due to the possibility of the presentation of specific tumor antigens and activation of the antitumor response in the patient's body.

In this regard, the preparation of functionally active DCs in vitro and the activation of their tumor-associated antigens to stimulate the cytotoxic response is one of the priorities in the development of antitumor vaccines based on dendritic cells, since it allows mobilizing the protective systems of the patient’s body using natural methods for recognizing tumor antigens and their subsequent elimination.

In view of the low content of dendritic cells in human blood, the standard scheme for the preparation of dendritic cell vaccines involves the differentiation of patient’s peripheral blood monocytes into dendritic cells under the influence of a set of cytokines (Adaptation of the method for culturing human dendritic cells from peripheral blood monocytes for clinical use, Chkadua G.Z. et al., Russian Biotherapeutic Journal, vol. 1, No. 3, 2002, pp. 56-62. Subsequently, dendritic cells obtained in a similar manner are loaded with a purified prep arata of a specific tumor antigen or a total lysate of tumor cells.

The most optimal method for loading tumor antigens is transduction of dendritic cells with antigen-containing viral vectors, which provide highly efficient presentation of antigens by molecules of the main histocompatibility complex of the first class (MHCI). As a rule, lentiviral vectors based on the human immunodeficiency virus type 1 HIV1 are used for transduction, which allow integrating the target sequence into the genomic DNA of the cell, thereby ensuring a constant and stable level of antigen expression.

In particular, cells constructed for expressing at least one cytokine and at least one antigen that induce self-differentiation of dendritic cell progenitor cells (DC) into functional antigen-representative induced DCs (iDCs) are known in the art. Particularly preferred is a lentiviral vector that has a mutant integrase with a nucleic acid sequence (WO 2014122035 (A2).

However, the use of lentiviral vectors to create dendritic cell vaccines is limited by the low transduction efficiency of dendritic cells.

Known recombinant plasmid DNA pCI-UB-POLYEPI, a prototype of the present invention, containing epitopes of tumor-associated antigens for colorectal cancer, and a method for its use to stimulate a specific antitumor immune response against colorectal cancer cells. The use of dendritic cells transfected with a DNA construct encoding the immunogenic epitopes of tumor-associated antigens CEA, EpCAM and MUC4 is also known, which is an effective way to stimulate the cytotoxic potential of mononuclear cells (RF patent No. 2507265).

The present invention is the creation of a genetic construct that allows more efficient to prepare on its basis dendritic and T-cell vaccines for the treatment of malignant neoplasms.

The problem is solved by a new plasmid construct containing regulatory elements of the lentiviral vector for transduction of peripheral monocytes and dendritic cells, as well as an expression cassette that provides polycistronic expression of transcription factors c-Myc and BMI1 under the control of the EF1 alpha cell promoter.

Thus, the invention provides a genetic construct based on an expression plasmid lentiviral vector that encodes a sequence of transcription factor c-Myc, a self-cleaving peptide T2A and a sequence of transcription factor BMI1, as well as a number of regulatory elements: 5 'and 3' LTR (long terminal repeat), Rev (RRE or Rev-response element), Env is the regulatory sequence that ensures the packaging of the viral genome into the structure of the viral particle with the formation of the virion, CPPT is the central polypurine tract, WPRE (woodchuck hepat itis virus posttranscriptional regulatory element) and the EF1 alpha promoter, designed to induce the proliferation of monocytes and dendritic cells derived from human peripheral blood. The invention is illustrated by the following graphic materials:

FIG. 1 - Lentiviral vector pLEF1a-tagRFP

FIG. 2 - Schematic representation of a genetic construct containing the sequence of transcription factors c-Myc and BMI1 for expression under the control of the cell promoter EF1a

FIG. 3 - Dynamics of changes in the number of dendritic cells transduced with the expression construct pLEF1a-cMyc-T2A-BMI1 and control samples during ex vivo cultivation.

The technical result of the invention is the provision of effective constitutive expression of transcription factors c-Myc and BMI1 construct, when introduced into peripheral monocytes and dendritic cells, these cell populations begin to show proliferative activity, and can be expanded during ex vivo cultivation. The invention is illustrated by the following examples:

Example 1. Obtaining a genetic construct

The lentiviral vector pLEF1a-tagRFP of the structure indicated in FIG. 1 is used as the basis for creating a genetic construct containing sequences of transcription factors c-Myc and BMI1. one.

The following nucleotide sequences are inserted into the presented vector by the method of cloning at restriction sites with rarely cleaved endonucleases:

Последовательность, кодирующая транскрипционный фактор с-Мус

Sequence of transcription factor c-Myc

SEQ ID No. 1

Последовательность саморасщепляющегося пептида Т2А

The sequence of self-cleaving peptide T2A

SEQ ID №2

SEQ ID No. 2

Последовательность, кодирующая транскрипционный фактор BMI1

The sequence encoding the transcription factor BMI1

SEQ ID No. 3

These sequences are introduced from the 3'-end from the promoter region of the elongation factor 1 alpha (EF1alpha).

The introduced sequences are generated by PCR from cDNA obtained by reverse transcription of mRNA isolated from the peripheral mononuclear cells of a healthy donor. After amplification of the sequences encoding transcription factors, bridge PCR is performed to obtain a fusion construct consisting of the c-Myc sequence, the subsequent self-cleaving T2A peptide, and the subsequent sequence of the transcription factor BMI1. The amplified fragments are separated during horizontal agarose gel electrophoresis. To clean DNA fragments, use the Clean Up Mini kit (Eurogen, Russia). The purified DNA fragment and the pLEF1a-tagRFP lentiviral vector are treated with restriction endonucleases XbaI and SpeI, as a result of which the sequence of the red fluorescent tagRFP protein is excised from the lentiviral vector, followed by another step in the purification of restriction mixtures on an agarose gel. At the final stage, the obtained genetic sequences and expression vector are ligated. The correct orientation of the insert in the final vector is controlled by diagnostic PCR with primers EF1a seq dir and BMI Spe stop rev. Analysis of the nucleotide sequence of the obtained genetic constructs is carried out by sequencing, which proved the success of the cloning. The result of the work was to obtain a genetic construct containing sequences of transcription factors c-Myc and BMI1 for expression under the control of the cell promoter EF1a; the structure of the genetic construct is shown in FIG. 2.

Example 2. The structure of the genetic construct containing the sequence of transcription factors c-Myc and BMI1 for expression under the control of the cell promoter EF1a

The claimed genetic construct includes sequences encoding transcription factors c-Myc and BMI1 as well as a self-cleaving peptide sequence that provides polycistronic expression of transcription factors under the control of one promoter; and a number of regulatory sequences that ensure efficient assembly of the lentiviral vector into pseudovirus particles and are necessary for the successful integration of the expression cassette into the cell genome.

Regulatory sequences

5' и 3' LTR (long terminal repeat) - длинные концевые повторы, последовательности ДНК, обеспечивающие внедрение вирусного генома в геном клетки-хозяина при участии фермента интегразы.

5 'and 3' LTR (long terminal repeat) - long terminal repeats, DNA sequences that ensure the introduction of the viral genome into the genome of the host cell with the participation of the integrase enzyme.

Rev (RRE или Rev-response element) - регуляторная последовательность, обеспечивающая транспортировку молекулы вирусной мРНК из ядра в цитоплазму, где происходит ее экспрессия.

Rev (RRE or Rev-response element) is a regulatory sequence that ensures the transport of a viral mRNA molecule from the nucleus to the cytoplasm, where its expression occurs.

Env - регуляторная последовательность, обеспечивающая упаковку вирусного генома в структуру вирусной частицы с образованием вириона.

Env is a regulatory sequence that provides the packaging of the viral genome in the structure of the viral particle with the formation of the virion.

сРРТ - центральный полипуриновый тракт - последовательность, которая способствует проникновению обратно-транскрибируемой вирусной ДНК в ядро клетки, обеспечивая тем самым заражение митотически неактивных клеток.

cppp - the central polypurine tract - a sequence that facilitates the penetration of reverse transcribed viral DNA into the cell nucleus, thereby providing infection of mitotically inactive cells.

WPRE (woodchuck hepatitis virus posttranscriptional regulatory element) - энхансер - последовательность, обеспечивающая формирование особой пространственной структуры трансгена и увеличивающая вероятность распознавания промоторного региона факторами траскрипции. Кроме того, увеличивает стабильность мРНК трансгена.

WPRE (woodchuck hepatitis virus posttranscriptional regulatory element) - enhancer - a sequence that provides the formation of a special spatial structure of the transgene and increases the likelihood of recognition of the promoter region by transcription factors. In addition, it increases the stability of the transgene mRNA.

Промотор EF1 alpha. Эффективный промотор, обеспечивающий высокий уровень экспрессии во многих типах клеток.

The promoter is EF1 alpha. An effective promoter that provides a high level of expression in many types of cells.

Expressed sequences

с-Мус - последовательность транскрипционного фактора Мус, кодируемого человеческим геном MYC.

c-Myc is the sequence of the transcription factor Myc encoded by the human gene MYC.

Т2А - 2А последовательность из вируса Thosea asigna, саморасщепляющийся пептид, необходимый для разделения аминокислотных последовательностей транскрипционных факторов сразу после трансляции.

T2A - 2A sequence from Thosea asigna virus, a self-cleaving peptide necessary for the separation of amino acid sequences of transcription factors immediately after translation.

BMI1 - последовательность транскрипционного фактора BMI1 из белков группы Polycomb.

BMI1 is the sequence of the transcription factor BMI1 from proteins of the Polycomb group.

Example 3. Comparison of the dynamics of the number of dendritic cells during cultivation after administration of the expression construct pLEF1a-cMyc-T2A-BMH relative to non-transduced DC

To determine the effect of the introduction of the expression construct pLEF1a-cMyc-T2A-BMI1 on the activation of proliferation of dendritic cells obtained from peripheral monocytes by differentiation by culturing in the presence of growth factors interleukin-4 and granulocyte-macrophage colony-stimulating factor, dendritic cells transduced with pLEF1a T-cA-T -BMI1, cultured for 4 weeks. As a control, dendritic cells not subjected to lentiviral transduction are cultured in parallel in the same amount and density. Once a week, the number of cells is counted using a luminescent substrate Celltiter-Glo 2.0. The dynamics of the number of dendritic cells transduced with the pLEF1a-cMyc-T2A-BMI1 expression construct and control samples is shown in FIG. 3.

The results obtained indicate the high efficiency of the expression construct pLEF1a-cMyc-T2A-BMI1 on the stimulation of proliferation of peripheral dendritic cells, which indicates the high practical value of pLEF1a-cMyc-T2A-BMI1 for obtaining large quantities of functionally active dendritic cells, which may be in demand for the preparation of dendritic cell and T-cell vaccines used for cell immunotherapy.

Claims (1)

A genetic construct for inducing the proliferation of peripheral monocytes and dendritic cells in vitro obtained from human blood, including sequences encoding transcription factors c-Myc and BMI1, as well as their binding self-cleaving peptide sequence from Thosea asigna T2A virus, providing polycistronic expression of transcription factors under control one EF1alpha promoter; obtained on the basis of the expression plasmid lentiviral vector pLEF1a-tagRFP of the structure shown in FIG. 1, which includes a number of regulatory sequences: 5 'and 3' LTR long terminal repeats, Rev dependent element (RRE or Rev-response element), Env - regulatory sequence, which provides the packaging of the viral genome in the structure of the viral particle with the formation of the virion, cpp - central polypurine tract, post-transcriptional regulatory element WPRE, promoter EF1alpha.

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