RU2650774C1 - Effective targets of rna interference in the genom of hiv-1 and dyser's substrates are used for their disorder - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to a fields of medical and molecular genetics, as well as to gene therapy, and concerns a set of six Dyser’s substrates. Presented kit includes six Dyser’s substrates that form six siRNAs in human cells: 5'AAAAAGCAUCAGAAAGAACCUCCAUUU 3' – A1 (7); 5'AAAUGGAGGUUCUUUCUGAUGCUUUUU 3' – A1 (8); 5'AGACAUAGUUAUCUAUCAAUACAUGGA 3' – A2 (9); 5'UCCAUGUAUUGAUAGAUAACUAUGUCU 3' – A2 (10); 5'AGGAGUAGUGGAGUCUAUGAAUAAGGA 3' – A3 (11); 5'UCCUUAUUCAUAGACUCCACUACUCCU 3' – A3 (12); 5'GUGUGGACUAUAGUAGGUAUAGAAUAU 3' – A4 (13); 5'AUAUUCUAUACCUACUAUAGUCCACAC 3' – A4 (14); 5'ACCAGGACAGACAUGGUAUGGAACAGG 3' – A5 (15); 5'CCUGUUCCAUACCAUGUCUGUCCUGGU 3' – A5 (16); 5'GUGCGAGAGCGUCAGUAUUAAGUGGGG 3' – A6 (17); 5'CCCCACUUAAUACUGACGCUCUCGCAC 3' – A6 (18), which when injected into human cells are capable to infect corresponding conservative targets in HIV-1 mRNA (A1–A6): 5'AAAAAGCATCAGAAAGAACCTCCATTT 3' – A1 (1); 5'AGACATAGTTATCTATCAATACATGGA 3' – A2 (2); 5'AGGAGTAGTGGAGTCTATGAATAAGGA 3' – A3 (3); 5'GTGTGGACTATAGTAGGTATAGAATAT 3' – A4 (4); 5'ACCAGGACAGACATGGTATGGAACAGG 3' – A5 (5); 5'GTGCGAGAGCGTCAGTATTAAGTGGGG 3' – A6 (6), located in the domains of reverse transcriptase (targets A1 and A2) and integrases (target A3) of the pol gene, in the mRNA of the vpu gene (target A4), as well as mRNA of the env gene in its domain gp120 (target A5) and mRNA of the gag gene in the p17 domain (target A6), and cause suppression of virus production in human cells.

EFFECT: invention can be used in medicine and scientific research.

1 cl, 2 dwg, 4 tbl, 1 ex

Description

The invention relates to the fields of medical and molecular genetics, as well as to gene therapy, and is associated with the identification of six effective targets for RNA interference in human immunodeficiency virus type 1 (HIV-1) subtype A. These targets are identified based on a detailed analysis of nucleotide sequences in vital important pol, vpu, env, and gag genes of HIV-1 encoding reverse transcriptase (RT), integrase (Int), vpu protein, gp120 protein, and p17 protein, respectively.

They are located in the most conservative 27-nucleotide regions of these genes. In addition, prototypes of drugs, Dyser substrates, which are 27-nucleotide double-stranded RNAs and effectively hit these targets in virus transcripts using RNA interference, have been proposed. In this case, transcripts are cut, as a result of which the corresponding genes are not expressed. This causes a break in the life cycle of HIV-1 and blocks its production in human cells.

HIV-1 reverse transcriptase (RT) synthesizes DNA copies on the RNA matrices of the virus, and integrase (Int) enables the integration of viral DNA into the chromosome of the host cell. These products are encoded by the pol gene.

The vpu protein is necessary for the isolation of daughter virions from the cell. In addition, it interacts with cellular factors, blocking the antiviral defense of the host.

The p17 matrix protein, which is encoded by the gag gene, contains a membrane localization domain (M, membrane targeting). This structural protein surrounding the capsid plays an important role at many stages of the virus's life cycle, including replication, targeting RNA to the plasma membrane, virion assembly, and several others [Fiorentini S, Marini E, Caracciolo S, Caruso A. Functions of the HIV-1 matrix protein p17. New Microbiol. 2006 Jan; 29 (1): 1-10].

The gp120 protein, which is encoded by the env gene, is required for the virus to enter the cell.

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., 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.

RNA interference is a powerful natural mechanism for regulating gene expression. It is triggered by cutting with a Dicer enzyme double-stranded RNA into 21-nucleotide fragments that have two nucleotide protrusions at the 3 'ends. Dicer facilitates loading of such RNAs (double-stranded siRNAs) into complex protein complexes - RISC - in which only the antisense strand of RNA (single-stranded siRNAs) remains. It serves as a guide through which RISC recognizes complementary nucleotide sequences in mRNA and cuts them. Thus, the expression of the corresponding gene is blocked. RNA interference mechanisms require complete complementarity between the target in the virus transcript and the corresponding siRNA [Pusch et al., Nucl. Acids Res., 2003, 31, 6444-6449; Senserrich et al., Virology, 2008, 372, 421-429]. HIV-1 is highly variable. That is why the creation of HIV / AIDS gene therapy technology requires a thorough study of RNA interference targets and selection of only such fully complementary targets that are identical in 70-95% of the clinical variants of viruses [Kretova OV, Chechetkin VR, Fedoseeva DM, Kravatsky YV, Sosin DV, Alembekov IR, Gorbacheva MA, Gashnikova NM, Tchurikov NA. Analysis of variability in HIV-1 subtype A strains in Russia suggests a combination of deep sequencing and multi-target RNA interference for silencing of the virus. AIDS Res Hum Retroviruses. 2016 Jul 30. Epub ahead of print DOI: 10.1089 / AID.2016.0088 PMID: 27476852].

Using capillary and deep sequencing of the HIV-1 subtype A genome in two cohorts of patients in Russia, variability was studied in six selected RNA interference targets. As a result, it was concluded that the selected six targets are highly conservative and quite suitable for use in gene therapy (Table 1).

Six Dyser substrates are proposed (Table 2), which are designed to effectively suppress the production of these clinical HIV-1 variants in human cells. They 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.

Closest to this invention is a genetic construct that allows the expression of two anti-HIV-1 siRNAs corresponding to targets A1 and A3 of this Application (RF patent No. 2552486, published May 6, 2015). In addition, the same targets A2, A4, A5 and A6 are shown in RF patent 2552607 (targets A1 and A6), published May 7, 2015; in the patent of the Russian Federation 2607381, published on January 10, 2017 (targets A1 and A2).

The present invention is characterized in that

1. It is proposed to use not two, but six of the above highly conservative RNA interference targets in the HIV, pol, vpu, env and gag genes identified in patients in Russia;

2. use not genetic constructs expressing 27-30-nucleotide RNA hairpins, but 27-nucleotide double-stranded RNAs, Dicer substrates.

The differences in the nucleotide sequences of the targets of this Application from the targets in earlier Applications and Patents are presented in Table 3.

It was previously described that 27-nucleotide double-stranded RNAs can enhance the efficiency of RNA interference by two orders of magnitude [Kim DH, Behlke MA, Rose SD, Chang MS, Choi S, Rossi JJ. Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy. Nat Biotechnol. 2005; Grimm D, Streetz KL, Jopling CL, Storm TA, Pandey K, Davis CR, Marion P, Salazar F, Kay MA. Fatality in mice due to oversaturation of cellular microRNA / short hairpin RNA pathways. Nature. 2006 May 5; 441 (7092): 537-41]. According to other data, the effectiveness of RNA interference, triggered by Dyser substrates, increases for different targets by 2-5 times [RF Patent No. 2385939, publ. 04/10/2010; Alembekov I.R., Kretova O.V., Churikov N.A. Analysis of genetic constructs expressing antiviral siRNAs in a non-viral test system. Questions of Virology, 2011, v. 56, No. 2, p. 32-35].

Data on HIV-1 subtype A RNA interference targets in the clinical variants of the virus isolated from patients in Russia were obtained by capillary and deep sequencing of six RNA interference targets and deposited in GenBank (accession numbers: KC681847-KC681888) and NCBI (Biosamples) : SAMN05823508 and SAMN05828325). All identified targets are characteristic of virus isolates in Russia and are found in 83-92% of viruses (Table 1). In addition, targets identical to them are found in HIV-1 isolates from countries other than Russia (Table 4). Identical targets were searched for in the current nucleotide sequence databases using the BLAST program (NCBI). The number of studied HIV-1 isolates in different countries can vary significantly. Nevertheless, the search data indicate that the identified targets in addition to Russia are also often found in HIV-1 isolates of 11-20 subtypes of different countries on all continents. In addition, targets identical to targets A1, A3 and A6 of this Application are found in recombinant HIV-1 AG viruses, which have been described previously [Baryshev, P.B., Bogachev, V.V. and Gashnikova, N.M. Genetic characterization of an isolate of HIV type 1 AG recombinan form circulating in Siberia, Russia. J. Arch. Virol. 157 (12), 2335-2341 (2012)].

The technical results of the invention are six conservative RNA interference targets (A1-A6) shown in Table 1, as well as six Dyser substrates corresponding to them, which are introduced into human cells using capillary and deep sequencing in HIV-1 isolates in Russia capable of affecting transcripts of the pol genes (reverse transcriptase, RT, and integrase domains, int), vpu, env (gp120 domain) and gag (p17 domain) of HIV-1 and cause suppression of virus production in human cells.

The presented technical result is achieved by the delivery of one or more Dyser substrates (Table 2) into human cells.

These Dicer substrates were tested using a non-viral system. This system is designed to quantify the biological activity of siRNAs, which are formed in vivo upon expression of genetic constructs or upon the introduction of double-stranded RNA. The system is based on the psiCHECK ™ -2 vector (Promega, GenBank sequence number AY535007). The vector contains the firefly luciferase gene (Photinus pyralis) and the coral luciferase gene (Renilla). These luciferases cause a luminescence of different wavelengths, and their expression can be measured using a luminometer. 27-nucleotide DNA sequences containing A1, A2, A3, A4, A5, A6 targets corresponding to the selected RNA interference targets in virus transcripts were inserted separately into the 3 'terminal non-coding region of the Renilla gene. These DNAs are obtained by chemical synthesis of DNA oligonucleotides. Table 1 presents the texts of the targets. DNA fragments containing the targets are cloned into 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 transfection of human culture cells. In this case, the DNA of the plasmid created on the basis of the psi-CHECK-2 vector is used, in which a DNA sequence containing six RNA interference targets indicated in Table 1 was cloned into the 3 ′ terminal non-coding sequence of the Renilla luciferase gene. with the indicated plasmid, chemically synthesized double-stranded RNA preparations (Dicer substrates) are listed in Table 2. They are a natural substrate of Dicer, an enzyme that cuts out 21 nucleotide duplexes of RNA from a hairpin. If Dyser operates on an RNA duplex of more than 23 pairs of nucleotides (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, there is an unwinding of siRNA chains and its release 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” that recognizes 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 firefly luciferase (557 nm) encoded by the same psiCHECK-2 DNA serves as an internal control and is used to normalize the data.

For the selection of the most conserved regions of HIV-1, multiple alignments of the virus sequences known in 2001 were performed. Then, in the most conservative areas, they searched for targets using on-line services designed to search for siRNAs. Eleven criteria were used to select sequences of RNA interference targets in mRNA of HIV-1 genes. Most of them meet the siRNA selection criteria of Dharmacon RNA Technologies, http://dharmacon.gelifesciences.com/design-center/.,skb. Thus, A2-A6 targets were selected. Target A1 was selected according to Tushl’s criteria (Tom Tuschl's rules, http://www.protocol-online.org/prot/Protocols/Rules-of-siRNA-design-for-RNA-interference--RNAi--3210.html) .

These criteria are important so that the siRNA antisense strand remains in the RISC complex. Each of the Dicer substrates shown in Table 2 in the genetic constructs meets at least ten of the eleven criteria listed below that were used to select core 19-nucleotide siRNA sequences (the corresponding regions are underlined in the target sequences shown in Table 1):

- 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].

The Dicer substrates shown in Table 2 provide a targeted effect on the mRNA of the HIV, 1 pol, vpu, env and gag genes. 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, conservative targets were identified and the corresponding Dyser substrates attacking six different targets in HIV-1 were obtained. Thus, a significant suppression of the production of the virus in human cells is caused, which is largely independent of the variability of the virus.

Brief description of tables and figures

Table 1 shows the 27-bp sequences corresponding to the identified targets for RNA interference in HIV-1. DNA sequences are presented in the orientation of 5'-3 '. In parentheses are the sequence numbers. Areas corresponding to core 19-nucleotide siRNA sequences are underlined. The percentage of viruses having this target in HIV-1 isolates in Russia is indicated. These 27 nucleotide RNA interference targets were cloned into psiCHECK-2 vector. Artificial XhoI and NotI sites that were used for cloning are not indicated.

Table 2. Sequences of 27-nucleotide plus and minus RNA chains that form Dyser substrates after pairwise annealing (sequence numbers are indicated in parentheses).

Table 3. Differences in the nucleotide sequences of RNA interference targets and genetic constructs from earlier Patents and Applications from the targets of this Application. Targets A1-A6 - in this Application are shorter by 1-3 nucleotides (shown in green in the texts of previous Patents and Applications). In addition, target A4 has a difference in one position (shown in yellow).

Table 4. Search for homologous DNA sequences of targets in complete non-redundant nucleotide databases of NCBI using the BLAST program. Set the display of 5000 sequences from the databases [Nucleotide collection (nr / nt)]. The third column of the table shows the found number of identical sequences for this target. A5 target was found only in Russia.

FIG. 1. 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. 2. Evaluation of the effectiveness of RNA interference in a non-viral test system. RNA interference is induced by the introduction of Dicer substrates into HEK293T cells transfected with plasmids based on the psiCHECK-2 vector and containing RNA interference targets (Table 1). The luminescence data of the Renilla luciferase gene upon co-transfection of “randomized” targets with Dicer substrates, targets without the addition of Dicer preparations, and targets together with the corresponding Dicer substrates are given. * - p <0.007. The experiments were performed in three parallels (n = 3).

The implementation of the invention

Example 1. Evaluation of the effectiveness of RNA interference caused by the created Dicer substrates in the non-viral system

On the eve of transfection (18–20 hours), HEK293 cells are seeded into the wells of a 24-well Nunc 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 transfection at room temperature as follows (per experimental point). To 200 μl of serum-free DMEM medium (SFM), 40 ng of DNA of the genetic construct in psiCHECK-2 vector containing RNA interference targets was added (see Table 3). 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 and incubated for 1 hour at 37 ° C in a CO ₂ incubator.

To introduce Dicer substrates into cells, annealing of pairs of complementary RNAs is first performed in a solution containing 10 mM tris-HCl, pH 7.4; 20 mM NaCl and 40 pmol of each of the RNAs (per point) by incubating the resulting solution in a water bath cooling from 65 ° C to 25 ° C for 20 minutes. SiTransfection Reagent (Santa Cruz) is used to transfect annealed RNA (Dicer substrate). First, two mixtures are prepared - 1 μl Dicer Substrate (40 pmol) in 50 μl SFM, and 2 μl siRNA Transfection Reagent in 50 μl SFM (per point). Then both solutions are mixed, incubated for 15-45 minutes at room temperature.

After which the medium above the cells is sucked off, one wash is carried out once with 100 μl of SFM. Then 400 μl of SFM is added to the mixture containing the Dicer substrate and siRNA Transfection Reagent, the mixture is slowly pipetted for mixing and added to the well. After incubation for 7 hours, a complete medium of 500 μl was added to the well and incubation continued for 24 hours.

Next, about 1 ml of medium above the cells is aspirated and 800 μl of complete medium warmed up to 37 ° C is added. Incubation is continued for another 48 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 1 × PBS, and the glowworm and firefly luciferase were measured according to the recommendations of the Dual-Luciferase Report kit er Assay System (Promega) on the Reporter Microplate Luminometer (Turner BioSystems). In FIG. 2 shows the results of testing genetic constructs in a non-viral system. It can be seen that Dyser substrates specifically inhibit target expression by 81-91%.

Effective targets of RNA interference in the HIV-1 genome and Dicer substrates designed to defeat them

Nucleotide DNA Sequences:

5 'AAAAAGCATCAGAAAGAACCTCCATTT 3' (1)

5 'AGACATAGTTATCTATCAATACATGGA 3' (2)

5 'AGGAGTAGTGGAGTCTATGAATAAGGA 3' (3)

5 'GTGTGGACTATAGTAGGTATAGAATAT 3' (4)

5 'ACCAGGACAGACATGGTATGGAACAGG 3' (5)

5 'GTGCGAGAGCGTCAGTATTAAGTGGGG 3' (6),

RNA nucleotide sequences:

5 'AAAAAGCAUCAGAGAAAGAACCUCCAUUU 3' (7)

5 'AAAUGGAGGUUCUUUCUGAUGCUUUUU 3' (8)

5 'AGACAUAGUUAUCUAUCAAUACAUGGA 3' (9)

5 'UCCAUGUAUUGAUAGAUAACUAUGUCU 3' (10)

5 'AGGAGUAGUGGAGUCUAUGAAUAAGGA 3' (11)

5 'UCCUUAUUCAUAGACUCCACUACUCCU 3' (12)

5 'GUGUGGACUAUAGUAGGUAUAGAAUAU 3' (13)

5 'AUAUUCUAUACCUACUAUAGUCCACAC 3' (14)

5 'ACCAGGACAGACAUGGUAUGGAACAGG 3' (15)

5 'CCUGUUCCAUACCAUGUCUGUCCUGGU 3' (16)

5 'GUGCGAGAGCGUCAGUAUUAAGUGGGG 3' (17)

5 'AC AC CCCC AC AC AC AC AC AC AC' '18 18 18 18 (18)

Claims (21)

A kit comprising six Dicer substrates, which are obtained by pairwise annealing of complementary RNA sequences: 5'AAAAAGCAUCAGAGAAAGAACCUCCAUUU 3 '- A1 (7) 5'AAAUGGAGGUUCUUUCUGAUGCUUUUU 3 '- A1 (8) 5'AGACAUAGUUAUCUAUCAAUACAUGGA 3 '- A2 (9) 5'UCCAUGUAUUGAUAGAUAACUAUGUCU 3 '- A2 (10) 5'AGGAGUAGUGGAGUCUAUGAAUAAGGA 3 '- A3 (11) 5'UCCUUAUUCAUAGACUCCACUACUCCU 3 '- A3 (12) 5'GUGUGGACUAUAGUAGGUAUAGAAUAU 3 '- A4 (13) 5'AUAUUCUAUACCUACUAUAGUCCACAC 3 '- A4 (14) 5'ACCAGGACAGACAUGGUAUGGAACAGG 3 '- A5 (15) 5'CCUGUUCCAUACCAUGUCUGUCCUGGU 3 '- A5 (16) 5'GUGCGAGAGCGUCAGUAUUAAGUGGGG 3 '- A6 (17) 5'CCCCACUUAAUACUGACGCUCUCGCAC 3 '- A6 (18), which, when introduced into human cells, are capable of hitting the corresponding conservative targets in HIV-1 mRNA (A1-A6): 5'AAAAAGCATCAGAAAGAACCTCCATTT 3 '- A1 (1) 5'AGACATAGTTATCTATCAATACATGGA 3 '- A2 (2) 5'AGGAGTAGTGGAGTCTATGAATAAGGA 3 '- A3 (3) 5'GTGTGGACTATAGTAGGTATAGAATAT 3 '- A4 (4) 5'ACCAGGACAGACATGGTATGGAACAGG 3 '- A5 (5) 5'GTGCGAGAGCGTCAGTATTAAGTGGGG 3 '- A6 (6), located in the reverse transcriptase (target A1 and A2) and integrase (target A3) domains of the pol gene, in the vpu mRNA (target A4), as well as the env mRNA in its gp120 domain (target A5) and gag mRNA in the p17 domain ( target A6), and cause inhibition of virus production in human cells.

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