RU2552486C2 - CARTRIDGE GENETIC CONSTRUCT EXPRESSING TWO BIOLOGICALLY ACTIVE siRNAS EFFECTIVELY ATTACKING SUBTYPE A HIV-1 TRANSCRIPTS TARGETED TO iRNA OF REVERSE TRANSCRIPTASE AND INTEGRASE, AND ONE siRNA TARGETED TO iRNA OF CCR5 GENE IN RUSSIAN PATIENTS - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medical and molecular genetics, as well as to gene therapy. What is described is a genetic construct producing siRNAs, which are inhibitors of type 1 subtype A human immunodeficiency virus replication. A cartridge genetic construct based on the GeneClip-U1-neo vector producing three siRNAs that are the inhibitors of type 1 human immunodeficiency virus replication and the human CCR gene. The construct anti-HIV-1R-anti-HIV-3R-anti-CCR5-8 contains a fragment having a length of 30 bp and corresponding to a conservative region of a reverse transcriptase domain of the subtype A HIV-1 genome in Russian patients, a fragment having a length of 30 bp and corresponding to a conservative region of an integrase domain of the subtype A HIV-1 genome in Russian patients, as well as a 19 bp fragment corresponding to the iRNA of CCR5 gene.

EFFECT: technical result of the presented invention is developing effective anti-HIV preparations based on interfering RNAs (siRNAs) produced in cells by means of the introduced cartridge genetic construct, containing palindromes aimed at producing three siRNAs, two of which are targeted on targets in clinical versions of the subtype A HIV-1 in Russian patients, and tested with the use of a nonviral system.

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Description

The invention relates to the fields of medical and molecular genetics, as well as to gene therapy and is associated with the use of two previously identified two 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 two targets using deep sequencing of the HIV-1 subtype A genome in patients in Russia, sequences of the corresponding siRNAs are proposed to effectively suppress the production of these clinical HIV-1 variants in human cells.

This cassette expresses two biologically active anti-HIV-1 siRNA, which leads to an attack of two targets in the transcripts of the virus and a powerful inhibition of its reproduction in human cells. The indicated cassette genetic construct express RNA hairpins, which 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 indicated 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-HIV ribozymes in a human T lymphoblastoid cell line. Hum. Gene Ther., 9., 621-628; Veres G., Junker U., Baker J., Barske C., Kalfoglou C., Ilves 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 are a genetic construct that provides siRNA expression within HIV-infected cells of the body (RF patent No. 2425150, "Cassette genetic construct that expresses three biologically active siRNA, effectively attacking transcripts of the human immunodeficiency virus and CCR5 gene using RNA interference" )

The present invention is characterized in that (1) in the present genetic construct in the middle is a hairpin encoding siRNA directed not to RT, but to Int; (2) the hairpin at the beginning of the construction, aimed at RT, contains two nucleotide substitutions corresponding to variants of this target in patients in Russia. Thus, the proposed construct expresses two siPHKs that target RT and Int targets in HIV-1 subtype A variants in patients in Russia, and one siPHK expresses on the mRNA of the human CCR5 gene encoding the cell co-receptor involved in penetration 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: KS681847-KS681888).

The technical result of the invention is the use of two previously identified targets of RNA interference in HIV-1 (RF patent 2385939) and one target in CCR5 mRNA (RF Patent 2425150) to suppress the production of virus variants in patients in Russia. This cassette genetic construct 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 cassette genetic construct based on the GeneClip ™ U1 Neomycin vector (Ac. Number AY 745746), http: //www.promega.eom/pnotes/89/l12416 21/12416 21.pdf.

The constructed anti-HIV-1R-anti-HIV-3R-anti-CCR5-8 genetic construct of 207 bp length contains the DNA insert shown in Table 1.

Table 1.

The anti-HIV-1R-anti-HIV-3R-anti-CCR5-8 cassette genetic construct expressing three biologically active siRNAs

Table 1 shows 19-30 bp sequences corresponding to transcripts of the virus or CCR5 mRNA. The lower case letters in bold in the following order indicate the regions corresponding to: the sense strand of the mRNA reverse transcriptase of the virus, its antisense strand; sense strand of mRNA Int virus, its antisense strand; sense chain mRNA of the CCR5 gene, its antisense chain. DNA sequences are presented in 5′-3 ′ orientation. The lowercase letters indicate the texts of loops and spacers containing artificial sites for restriction enzymes EcoRI or BamHI (bold lowercase letters). Loops are formed in the transcript after annealing the complementary regions of the palindromes. 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. This design is designed to defeat two regions of mRNA encoding conserved regions of reverse transcriptase (RT) and integrase (Int) of the virus and 5 ′ region of the human CCR5 gene mRNA in the region of the first codons. The nucleotide substitutions in the RT and Int targets, characteristic for HIV-1 subtype A in patients in Russia, are obscured.

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

Table 2.

Target RNA interference anti-HIV-1R and anti-HIV-3R in patients in Russia, corresponding to the regions of RT and Int

In targets directed to reverse transcriptase and integrase mRNA, differences were found that are characteristic of the virus genome in patients in Russia (two positions of nucleotide substitutions are indicated by shading). 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 lucifezases cause 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 virus transcripts were inserted. Domains were obtained by chemical synthesis of DNA oligonucleotides. Table 2 presents the texts of the domains that were cloned in the psiCHECK ™ -2 vector at the XhoI and NotI restriction enzyme sites.

Table 3.

RNA interference targets cloned in psiCHECK-2 vector to test its effectiveness in the non-viral system

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 (targets HIV-1R and HIV-3R) 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.

Testing the biological activity of the cassette is carried out in experiments on co-transfection of human culture cells. Two DNA preparations are used: a cassette genetic construct containing the U1 RNA polymerase II promoter and coding for three siPHKs, as well as a DNA plasmid based on the psi-CHECK-2 vector containing three cloned domains corresponding to these siPHKs (RNA targets interference) (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 duplex RNAs from hairpins. If Dicer works on an RNA duplex of more than 19 pairs of nucleotides in length (a natural substrate), then, remaining connected with the double-stranded siRNA, it 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 insertions in the genetic constructs shown in Table 1 corresponds to 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,

- the absence of G at position 13 of the sense chain,

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

- localization of the target 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.

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

Figure 2 shows the physical map of the vector psiCHECK-2.

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

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 24-well Nunc tablet — 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). To 200 μl of serum-free DMEM medium (SFM), 100 ng of the DNA of the genetic construct in the GeneClip-U1-neo 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 are added, and the cell suspension is completely transferred to 0.5 ml Eperdorf 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 luminescent firefly and Renilla fluorescence was measured according to the recommendations for the Dual-Luciferase Reporter A kit ssay System (Promega) on the Reporter Microplate Luminometer (Turner BioSystems). Figure 3 shows the results of testing genetic constructs on a non-viral system. It is seen that genetic constructs specifically suppress target expression by 60-96%.

Brief Description of the Drawings

Figure 1 shows the physical map of the vector, which was used to create a cassette genetic construct that causes RNA interference directed against transcripts of the virus and CCR5 mRNA. Ampicillin resistance gene (Amp), polyadenylation site and SV40 enhancer are indicated.

Figure 2 shows the physical map of the vector that was used in the non-viral system to quantify the effectiveness of RNA interference caused by the generated cassette genetic construct. Ampicillin resistance gene (Amp), polyadenylation site and SV40 enhancer are indicated.

Figure 3 presents the test data of the design in a non-viral system. Data are presented in% on the effect of the anti-HIV-1R-anti-HIV-3R-anti-CCR5-8 genetic construct to the original GeneClip-U1-neo vector that does not contain an insert or contains a “randomized” sequence of targets; HIV-1R is the target corresponding to the RT region in patients in Russia; HIV-3R — target corresponding to the Int region in patients in Russia; 1R-3R-CCR5 are targets for the two above siRNAs and for siRNA directed to CCR5 mRNA.

Claims (1)

The cassette genetic construct of anti-HIV-1R-anti-HIV-3R-anti-CCR5-8 based on the GeneClip-U1-neo vector containing the insert shown in Table 1 producing three siRNA inhibitors of the reproduction of human immunodeficiency virus type 1, subtype A, and human CCR5 gene expression - and containing two fragments of the HIV-1 genome of subtype A, detected in patients in Russia, corresponding to conservative regions of the domain of reverse transcriptase and integrase, as well as the 19 bp region corresponding to the CCR5 gene (shown in the third line):

where lowercase letters indicate the areas of loops between the parts of the palindromes, capable of forming double-spiral duplexes.

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