RU2630644C1 - Cartridge genetic construct expressing two biologically active sirnas effectively attacking targets in mrna of vpu and env genes of hiv-1 subtype a in russian patients, and one sirna aimed at ccr5 gene mrna - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: proposed anti-HIV-4R-anti-HIV-5R-anti-CCR5-8 cartridge genetic construct is derived from the GeneClip-U1-neo vector and produces three siRNA inhibitors of human immunodeficiency virus type 1 and human CCR5 gene. The construct contains a 28 bp fragment corresponding to the conservative region of the vpu gene of HIV-1 subtype A in Russian patients (first line), a 30 bp fragment corresponding to the conservative region of the env gene (its GP120 domain) of HIV-1 subtype A in Russian patients (second line), and a 19-bp fragment corresponding to the CCR5 gene mRNA (third line): 5'TGTGTGGACTATAGGTATAGAATGTttcaagagaACATTCTATACCTACTATAGTCCACACA gcgaattctgACCAGGACAGACATGGTATGGAACAGGTGAagagaattcTCACCTGTTCCATACCATGTCTGTCCTGGTatggatcctcGGAACAAGATGGATTATCActtcctgtcaTGATAATCCATCTTGTTCC 3'. Lowercase letters show the regions of loops between parts of palindromes capable of forming double-helix duplexes.

EFFECT: invention provides a targeted effect on the mRNA of HIV-1 subtype A variants in Russia with the two appropriate siRNAs, as well as on the CCR5 mRNA, which leads to suppression of their reproduction.

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Description

The invention relates to the fields of medical and molecular genetics, as well as to gene therapy, and involves the use of previously identified targets for RNA interference in human immunodeficiency virus type 1 (HIV-1) subtype A to create a cassette genetic construct. Based on a detailed analysis of the nucleotide sequence in the vpu gene, which encodes a protein that promotes the isolation of daughter virions from the cell, and the env gene, which encodes the protein necessary for the virus to enter the cell, by deep sequencing of the HIV-1 subtype A genome in patients in Russia, the following sequences are proposed the corresponding two siRNAs designed to effectively suppress the production of these clinical HIV-1 variants in human cells.

The target in the env gene attacks the mRNA region encoding the GP120 glycoprotein. This protein has a CD4 cell receptor binding site and seven transmembrane domains of chemokine receptors serving as co-receptors for HIV-1. The target in the vpu gene attacks the 3 'coding region of the mRNA encoding the viral protein U (Vpu).

This cassette expresses two biologically active anti-HIV-1 siRNA, which leads to the attack of two targets in the transcripts of the virus encoding the vital proteins Vpu and ENV, and a powerful inhibition of its reproduction in human cells. Said cassette genetic construct expresses RNA hairpins that are processed in vivo into biologically active siRNAs. The latter are efficiently delivered to protein complexes that cut virus transcripts, thereby inhibiting its reproduction. In addition, this cassette expresses the biologically active siRNA attacking the mRNA of the human CCR5 gene. The specified gene encodes a co-receptor, facilitating the penetration of the virus into human T-lymphocytes. Such a genetic construct can be used in medicine for gene therapy of AIDS and HIV infection, as well as in scientific research for the powerful suppression of the production of HIV-1 subtype A.

Currently, the main approach to treating AIDS and HIV infection is the appointment of antiviral chemotherapy for patients, including drugs that act on the key HIV-1 enzymes - reverse transcriptase, integrase, protease. However, despite the large number of developed drugs, there is a problem of the effectiveness of the applied antiviral therapy. The main reason for the treatment failure is adaptive mutagenesis of the virus, leading to the emergence of virus variants that are resistant to antiviral drugs. Toxicity and the high cost of the drugs used are also serious problems.

Known use of ribozymes and antisense RNA for the treatment of HIV infection [Dorman N. And Lever A.M. (2001) RNA-based gene therapy for HIV infection. HIV Med., 2, 114-122; Michienzi A., Conti L., Varano B., Prislei S., Gessani S., and Bozzoni I. (1998) Inhibition of human immunodeficiency virus type 1 replication by nuclear chimeric anti-HIVribozymes in a human T lymphoblastoid cell line. Hum. Gene Ther., 9., 621-628; Veres G., Junker U., Baker J., Barske C., Kalfoglou C., lives H., Escaich S., Kaneshima H., and Bohnlein E. (1998) Comparative analysis of intracellularly expressed antisense RNAs as inhibitors of human immunodeficiency virus type 1 replication. J. Virol., 72, 1894-1901], which block several HIV genes, which reduces its activity.

Closest to this invention is a genetic construct that provides siRNA expression inside HIV-infected cells of the body (RF patent No. 2425150, published November 23, 2009).

The present invention is characterized in that in the present genetic construct, in the first and second positions there are studs encoding two siRNAs directed to mRNAs encoding Vpu and GP120, respectively. The hairpin regions contain nucleotide substitutions that are characteristic of variants of these targets in patients in Russia (Table 1). 6 such substitutions were found for the vpu mRNA region and 13 for gp120 mRNA region. Thus, the proposed construct expresses two siRNAs that target targets in mRNA encoding Vpu and GP120 in HIV-1 subtype A variants in patients in Russia, and one siRNA - on mRNA of the human CCR5 gene encoding a cell co-receptor, involved in the penetration of the virus into T-lymphocytes. Data on HIV-1 subtype A RNA interference targets in the clinical variants of the virus isolated from patients in Russia were obtained using capillary and deep sequencing and deposited in GenBank (accession numbers: KC681847-KC681888).

The technical result of the invention is a cassette genetic construct that contains three palindromes designed to form RNA hairpins. Expression of this construct in human cells leads to the formation of three interfering RNAs (siRNA). The proposed genetic construct encodes biologically active siRNAs that were selected using a non-viral test system using human cell cultures.

The technical result presented is achieved by creating a genetic cassette construct based on the GeneClip ™ U1 Neomycin vector (Ac.number AY745746), http://www.promega.com/pnotes/89/12416_21/12416_21.pdf.

The generated anti-HIV-4R-anti-HIV-5R-anti-CCR5-8 genetic construct of 202 bp length contains the DNA insert shown in Table 1.

Table 2 shows the differences in virus targets from the database — HIV-1 subtype A, GenBank sequence number AF316544, 1997 isolate HIV-1 subtype A from Congo; and in clinical variants of the subtype A virus isolated from patients in Russia.

In the RNA interference targets located in the mRNA of the vpu gene and env gene (in its GP120 domain) differences were found that are characteristic of the virus genome in patients in Russia (shading indicates 6 positions of nucleotide substitutions characteristic of the vpu gene and 13 characteristic of the env gene in his GP120 domain for subtype A virus variants in Russia). It is known that even single differences between the target and siRNA can significantly weaken the efficiency of RNA interference. Therefore, knowledge of the nucleotide sequence in the targets of the clinical variants of HIV-1 subtype A allows you to create genetic constructs that effectively affect the transcripts of the virus and its production in patients.

The indicated genetic construct was tested using a non-viral system. This system is designed to quantify the biological activity of siRNAs, which are formed in vivo during the expression of genetic constructs. It is based on the psiCHECK ™ -2 vector (Promega, GenBank sequence number AY535007). The vector contains the firefly luciferase gene and the coral luciferase gene (Renilla). These luciferases cause a luminescence of different wavelengths, and their expression can be measured using a luminometer. In the 3 'terminal non-coding region of the Renilla gene, short regions of the virus genome (domains) corresponding to the selected RNA interference targets in the virus transcripts were inserted. Domains were obtained by chemical synthesis of DNA oligonucleotides. Table 3 presents the texts of the domains that were cloned in the psiCHECK ™ -2 vector at the XhoI and NotI restriction enzyme sites.

Testing the biological activity of the cassette is carried out in experiments on co-transfection of human culture cells. In this case, two DNA preparations are used: a cassette genetic construct containing the U1 RNA polymerase II promoter and encoding three siPHKs, as well as a plasmid DNA based on the psi-CHECK-2 vector containing three cloned domains corresponding to three RNA interference targets) ( Table 3). In human cells, an insert containing palindromes is expressed on genetic constructs. The synthesized RNA forms hairpins, which are a natural substrate of Dicer, an enzyme that cuts out 21 nucleotide duplexes of RNA from a hairpin. If Dicer works on an RNA duplex of more than 19 pairs of nucleotides (a natural substrate), then it, remaining bound to the double-stranded siRNA, also actively participates in its “loading” into the RISC protein complex. In this complex, the siRNA chains are “untwisted” and released from one of them (from the so-called “passenger” chain). If the antisense siRNA remains in the complex, then it is used as a “gunner,” recognizing a complementary target in cell transcripts. After the complex is bound to the corresponding mRNA, it is cut by one of its proteins, which leads to the expression of the corresponding virus gene being turned off and its reproduction in the host cell being disrupted. In transfected cells, Renilla mRNA containing the test target of RNA interference in the 3'-non-coding region is cut, which leads to a sharp decrease in the blue glow caused by this luciferase (478 nm). The yellow-green glow caused by the firefly luciferase Photinus pyralis (557 nm) encoded by the same psiCHECK-2 DNA serves as an internal control.

In this study, eleven criteria were used to select sequences of RNA interference targets in the mRNA of the CCR5 gene. They are important in order for the siRNA antisense chain to remain in the RISC complex. Each of the inserts in Table 1 in the genetic constructs meets at least ten of the eleven criteria listed below that were used to select the siRNA core 19 nucleotide sequences:

- composition G / C 30-52%

- at least 3 A / U in positions 15-19 of the sense circuit

- lack of internal repetitions

- And in position 19 of the sense chain

- And in position 3 of the sense chain

- U at position 10 of the sense chain

- lack of G / C at position 19 of the sense chain

- lack of G at position 13 of the sense chain

- the absence of homopolymer paths (A) _4-5 or (T) _4-5 in the sense chain

- target localization in the conservative region of the virus genome

- lack of homology with known transcripts of the human genome.

Some of the above criteria for the selection of biologically active siRNAs are published [Reynolds A., Leake D., Boese Q., Scaringe S., Marshall W.S., Khvorova A. (2004) Rational siRNA design for RNA interference. Nature Biotechnol. 22, 326-330] and is available at http://www.dharmacon.com.

The cassette genetic construct shown in Table 1 provides a targeted effect on the mRNA of virus variants in Russia using the corresponding two siRNAs, as well as on CCR5 mRNA. The therapeutic result of this effect is the suppression of the reproduction of HIV-1 subtype A variants in patients in Russia.

The first type of human immunodeficiency virus is characterized by the rapid onset and selection of mutant variants that are resistant to various antiviral drugs. To solve the problem of viral variability, a cassette genetic construct was obtained that attacks different targets and therefore is largely independent of the variability of the virus.

Brief description of tables and figures

Tables 1-3 show the nucleotide sequences of DNA (numbers 1-4): a cassette construct that has a length of 202 bp and encodes three siRNAs (number 1); RNA interference targets in the mRNA of the vpu and gp120 genes detected in patients in Russia (numbers 2 and 3); RNA interference targets used for testing in the non-viral system (number 4).

In fig. Figure 1 shows a physical map of the GeneClipU1-neo vector.

In fig. 2 shows a physical map of the psiCHECK-2 vector.

In fig. 3 presents the results of testing the effectiveness of RNA interference caused by the created cassette genetic construct on a non-viral test system.

Table 1 shows the 19-30-bp sequences corresponding to the transcripts of the virus or CCR5 mRNA. Lower case letters in bold in the following order indicate: regions corresponding to DNA hairpins that correspond to sequences of sense and antisense chains of mRNA of the vpu, env virus genes, as well as the CCR5 gene (regions of sense mRNA are underlined). DNA sequences are presented in the orientation of 5'-3 '.

The lowercase letters indicate the texts of loops and spacers containing artificial sites for restriction enzymes EcoRI or BamHI (bold lowercase letters). Three loops are formed in the transcript after annealing the complementary regions of palindromes (semantic and antisense). The texts of the genetic construct correspond to the targets in the HIV-1 subtype A strains isolated from patients in Russia. The numbering of the CCR5 gene region is indicated by the sequence GenBank - U54994. The indicated design is intended to damage two regions of mRNA encoding the conserved regions of the vpu and env virus genes, as well as the 5 ′ region of the human CCR5 gene in the region of the first codons. Six nucleotide substitutions in the vpu target and thirteen in the env target, characteristic of HIV-1 subtype A in the patients in Russia, are obscured (only regions corresponding to sense RNAs are obscured). The number of this 202-bp DNA nucleotide sequence is indicated in parentheses.

Table 2. The numbers of DNA nucleotide sequences are indicated in parentheses.

In Table 3, lowercase letters indicate regions containing the restriction sites of the endonucleases XhoI and NotI, as well as two spacers between fragments of the HIV-1 genome (HIV-4R and HIV-5R targets) and CCR5 cDNA. Bold areas of the genome of the clinical variants of the virus in Russia and CCR5 cDNA that correspond to RNA interference targets are shown. The number of the given DNA nucleotide sequence is indicated in parentheses.

Fig. 1. Physical map of the vector GeneClip-U1-neo. The vector was used to create a cassette genetic construct causing RNA interference directed against virus and CCR5 mRNA transcripts. Ampicillin resistance gene (Amp), polyadenylation site and SV40 enhancer are indicated.

Fig. 2. Physical map of the psiCHECK-2 vector. The vector was used to create a non-viral system for quantifying the effectiveness of RNA interference caused by the generated cassette genetic construct. Ampicillin resistance gene (Amp), polyadenylation site and SV40 enhancer are indicated.

Fig. 3. The effectiveness of RNA interference caused by the cassette genetic construct anti-HIV-1R-anti-HIV-2R-anti-CCR5-8 in a non-viral test system for different targets cloned in the GeneClip-U1-neo vector. Data are presented in% on the effect of the anti-HIV-4R-anti-HIV-5R-anti-CCR5-8 genetic construct on the original GeneClip-U1-neo vector, which does not contain an insert or contains a “randomized” sequence of targets; HIV-4R is the target corresponding to the region of the vpu gene in patients in Russia; HIV-5R is a target corresponding to another region of the env gene (its domain encoding GP120) in patients in Russia; 4R-5R-CCR5 are targets for the two above-mentioned siRNAs and for siRNA directed to mRNA of the CCR5 gene. * - p <0.05. The experiments were performed in three parallels (n = 3).

The implementation of the invention

Example 1. Description of the genetic construct and method for its preparation

The GeneClipU1-neo vector is registered in GenBank (Accession # AC AY745746), has a size of 4,758 kb (kilobases - one thousand base pairs) and is characterized in that it contains:

R7 polymerase T7 start site: 1

U1 human RNA polymerase II promoter: 46-438

10 bp spacer: 439-448

U1 terminator: 449-465

RNA polymerase promoter SP6: 527-546

early enhancer / promoter of the SV40 virus: 798-1216

SV40 virus replication start signal: 1114-1179

neo gene: 1251-2045

synthetic poly (A): 2080-2128

β-lactamase gene: 3080-3940

T7 polymerase RNA promoter: 4742-3.

Chemically synthesized DNA oligonucleotides that correspond to genetic constructs producing RNA hairpins are inserted into this vector, which is supplied by Promega in a linear form (Table 1).

The psiCHECK-2 vector is registered in GenBank (Accession # AY535007), has a size of 6.27 kb and is characterized in that it contains:

early enhancer / promoter of the SV40 virus: 7-425

chimeric intron: 489-621

T7 polymerase RNA promoter: 666-684

Relilla luciferase gene: 694-1629

polylinker: 1636-1680

synthetic poly (A): 1688-1736

HSK-TK promoter: 1744-2496

firefly luciferase gene: 2532-4184

late signal poly (A) SV40: 4219-4440

β-lactamase gene: 4587-5447.

In this vector, chemically synthesized DNA oligonucleotides that correspond to the selected RNA interference targets are inserted into the XhoI and NotI sites of the polylinker (Table 2).

Example 2. Evaluation of the effectiveness of RNA interference caused by the created genetic constructs in the non-viral system

On the eve of co-transfection (in 18–20 hours), HEK293 cells are seeded into the wells of a Nunc 24-well plate — 50 thousand cells per 500 μl of DMEM (PanEco) culture medium containing glutamine and serum (complete medium) per well. On the day of the experiment, DNA samples are prepared for co-transfection at room temperature as follows (per experimental point). In 200 μl of serum-free DMEM medium (SFM), 100 ng of the DNA of the genetic construct in the GeneClip-U1-peo vector (see Table 1) and 10 ng of plasmid DNA containing the corresponding cloned domains in the psiCHECK-2 vector (see Table 2). After mixing, add 1 μl of freshly thawed TransFast reagent (Promega), shake the mixture and incubate for 15 minutes. Then the medium is aspirated from the wells with “sitting” cells, the tube with the DNA-TransFast mixture is shaken and the mixture is immediately added to the wells with cells. After an incubation of 1 hour at 37 ° C in a CO ₂ incubator, 600 μl of serum medium are added to the wells, the plate is stirred and incubated for 48-72 hours.

To measure luciferase expression, the medium above the cells in the wells of the plate is aspirated, 100 μl of trypsin-EDTA (PanEco) solution are added per well, after 10 min of incubation at 37 ° C in a CO ₂ incubator, 100 μl of complete medium is added, the cell suspension is completely transferred to 0.5 ml eppendorf tube, precipitated by centrifugation at room temperature in an Eppendorf centrifuge (5 min - 2000 rpm), the supernatant was aspirated, the cells were suspended in 70 μl of 1xPBS and the glowworm and firefly luciferase were measured according to the recommendations of the Dual-Luciferase Reporter kit Assay System (Promega) on the Reporter Microplate Luminometer (Turner BioSystems). In fig. Figure 3 shows the results of testing genetic constructs on a non-viral system. It is seen that genetic constructs specifically inhibit target expression by 77-90%.

Claims (3)

The cassette genetic construct anti-HIV-4R-anti-HIV-5R-anti-CCR5-8 based on the GeneClip-U1-neo vector containing an insert expressing three RNA hairpins encoding siRNA - inhibitors of the reproduction of human immunodeficiency virus type 1 subtype A and expression of the human CCR5 gene - and containing two fragments of the HIV-1 genome of subtype A, detected in patients in Russia, and corresponding to two conservative regions of the Vpu and GP120 domains (plus and minus the texts of palindromes corresponding to these regions are shown in the first and second lines) as well as the 19-bp region corresponding to the CCR5 gene ( plus and minus texts of this palindrome are given in the third line): 5'TGTGTGGACTATAGTAGGTATAGAATGTttaagagaACATTCTATACCTACTATAGTCCACACAgcgaattctgACCAGGACAGACATGGTATGGAACAGGTGAagagaattcTCACCTGTTCCATACCATGTGTGTGTGTGTGATGTGTGATCGATCGATCGATCATCGTATCTATCTATCAT where lowercase letters indicate the areas of loops between the parts of the palindromes, capable of forming double-spiral duplexes.

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