RU2385939C1 - Genetic makers attacking six new rna interference targets in transcripts of human immunodeficiency virus type 1 and suppressing virus reproduction in human cells - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: genetic makers of siSTRIKE-neo vector producing interfering RNA (siRNA), are inhibitors of reproduction of human immunodeficiency virus type 1. Invention allows producing effective anti-HIV preparations of siRNA produced in cells by the administered genetic makers containing palindrome intended for formation of siRNA production and selected with using non-virus and virus models.

EFFECT: invention can be used in medicine and researches.

12 cl, 3 dwg, 3 tbl, 3 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 new targets for RNA interference in type 1 human immunodeficiency virus (HIV-1) and the creation of genetic constructs, the expression of which leads to attack of these targets in virus transcripts and potent inhibition of its reproduction in human cells. These genetic constructs express 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. Such genetic constructs, as well as their corresponding double-stranded siRNA, can be used in medicine for gene therapy of AIDS and HIV infection, as well as in scientific research.

Currently, the main approach to treating AIDS and HIV infection is the appointment of antiviral chemotherapy for patients, which includes 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 S., Ilves H., Escaich S., Kaneshima H., and Bohnlein E. (1998) Comparative analisis 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 genetic constructs that ensure the expression of siRNA inside HIV-infected cells of the body (Application for US patent No. 20035999944, IPC C12N 15/867, publ. 03/27/2003). However, the effect of interfering RNAs produced in these published genetic constructs was evaluated using molecular clones of the human immunodeficiency virus, which does not allow predicting their effect on the natural pool of the viral population, which obviously has genetic diversity, which does not allow a more objective assessment of the antiviral effectiveness of such genetic constructions.

The technical result of the invention is to identify new targets for RNA interference in the virus and to obtain more efficient genetic constructs containing palindromes designed to form RNA hairpins. The expression of such constructs in human cells leads to the formation of interfering RNAs (siRNA). The proposed genetic constructs encode biologically active siRNAs that were selected using two test systems, non-viral and viral, using human cell cultures and various strains of HIV-1.

The technical result presented is achieved by creating eight genetic constructs based on the psiSTRIKE ^TM Neomycin Vector (AC AY497508). These genetic constructs contain DNA inserts shown in table 1.

In Table 1, 18-27-nucleotide regions corresponding to the sense strand of the virus RNA are shown in the row on the left, and the antisense region on the right. DNA sequences are presented in 5'-3 'orientation. The lowercase letters indicate the text of the loop formed in the transcript after annealing of its complementary regions, as well as the short region (dT) ₅ at the 3 'end of the construct, which is a terminator for RNA polymerase III. The texts of the genetic constructs correspond to the HIV-1 subtype A strain (HIV-1 subtype A), the GenBank sequence number is AF316544. The first six genetic constructs have 18-23 nucleotide regions corresponding to the sense strand of the RNA of the virus. The following six genetic constructs (No. 7-12 in Table 1) correspond to the same six regions in the sense strand of the RNA of the virus, but have lengths of 27 nucleotides. These constructs are intended to damage mRNA regions encoding conserved regions of reverse transcriptase (RT), integrase (Int), viral transmembrane protein U (Vpu), coat protein GP120, and matrix protein P17.

These genetic constructs were 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 ^TM -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 the 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 for the restriction enzyme saigas Xhol and Notl.

Table 2 in lowercase shows the regions containing the restriction sites of the endonucleases XhoI and NotI. The right column shows the regions of the virus genome that correspond to 27-30 nucleotide targets of RNA interference in the sequence AF316544.

Testing the biological activity of siRNA is carried out in experiments on co-transfection of human culture cells. In this case, two DNA preparations are used: a genetic construct containing the U6 promoter of RNA polymerase III and encoding a specific siRNA, as well as psi-CHECK-2 DNA containing a cloned domain corresponding to a given siRNA (target RNA interference). In human cells, an insert containing a palindrome is expressed on genetic constructs. The synthesized RNA forms a hairpin, which is a natural substrate of Dicer, an enzyme that cuts out 19-23 nucleotide duplexes of RNA from a hairpin. If Dyser operates on an RNA duplex of more than 19 pairs of nucleotides (a natural substrate), then, remaining connected with the double-stranded siRNA, they also actively participate in its “loading” into the RISC protein complex. In this complex, the siPHK chains are “untwisted” and released from one of them (from the so-called “passenger chain”). If the antisense siPHK remains in the complex, it is used as a “gunner” that recognizes a complementary target in the cell transcripts. After binding of the complex with the corresponding mRNA, it is cut by one of its proteins, which results in the expression of the corresponding gene of the virus being turned off and its reproduction in the host cell being disrupted. In transfected cells, Renilla mRNA containing in the 3'-non-coding region of the target of RNA interference 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 luciferase of firefly 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. They are important in order for the siRNA antisense chain to remain in the RISC complex. In addition, it was important to select targets less susceptible to adaptive mutagenesis and be able to use the potent human U6 RNA polymerase promoter. Each of the insertions in the genetic constructs given 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

- 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 presented on the website http://www.dharmacon.com.

The genetic constructs listed in Table 1 provide a targeted effect on the mRNA of the virus in human cells using siRNA produced in the cells. The therapeutic result of this effect is the suppression of HIV-1 reproduction in human cells.

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 series of genetic constructs was obtained, each of which stably expresses a specific siRNA.

To assess the effectiveness of the created genetic constructs, a viral model is also used - a human cell culture infected with various strains of HIV-1, in particular strains reproducing as acute and chronic infections. This approach allows conducting studies close to the real processes of HIV infection in the body. The use of laboratory strains of HIV-1 (in contrast to the molecular clones of the virus in the prototype) makes it possible to evaluate the effect of interfering RNA on the natural pool of the viral population, which obviously has genetic diversity. In addition, the viral model for testing the biological activity of siRNA allows the selection of not only effective siRNA, but also sterically accessible targets in virus transcripts. Biologically active (according to the above-described non-viral system) siRNA may be inactive when tested on the viral system, as in the latter case, the target RNA domain is in a different context of RNA sequences. In addition, it can be associated with proteins involved in the virus propagation cycle, which also makes this target in viral mRNA inaccessible to the RISC complex, a “charged” potentially active antisense siRNA. It also allows for a more objective assessment of the antiviral efficacy of genetic constructs.

Figure 1 shows the physical map of the vector psiSTRIKE-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 genetic constructs on a non-viral test system.

Example 1. Description of genetic constructs and method for their preparation

The psiSTRIKE-neo vector is registered in GenBank (Accession # AC AY497508), has a size of 4.56 kb (kitobases - one thousand base pairs) and is characterized in that it contains:

R7 polymerase T7 start site: 1

U6 human RNA polymerase III protector: 16-275

RNA polymerase RNA polymerase III human: 276

RNA polymerase promoter SP6: 329-348

start site RNA polymerase SP6: 331

early enhancer / promoter of the SV40 virus: 600-1018

SV40 virus replication start signal: 916-981

neo gene: 1053-1847

synthetic poly (A): 1882-1930

β-lactamase gene: 2882-3742

RNA polymerase T7 promoter: 4544-3.

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). In 200 μl of serum-free DMEM medium (SFM), 100 ng of the DNA of the genetic construct in the siSTRIKE-neo vector (see Table 1) and 10 ng of plasmid DNA containing the corresponding cloned domain 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 mixed 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 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 the glowworm and firefly luciferase were measured according to recommendations of the Dual-Luciferase Reporter Assay 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%.

Example 3. Suppression of virus reproduction in a MT-4 cell culture by introducing genetic constructs into cells using electroporation

Studies are carried out on the transplanted line of lymphoid cells MT-4. Cells were cultured on RPMI-1640 medium supplemented with 10% fetal bovine serum pre-inactivated by heating at 56 ° C for 30 minutes, 300 mg / ml L-glutamine and 100 μg / ml gentamicin.

The production and isolation of plasmid DNA is carried out using ammonium acetate according to the developed method for preparing DNA for transfection of mammalian cells [Saporito-Irwin S.M., Geist R.T. and Gutmann D.H. (1997) Ammonium Acetate Protocol for the preparation of plasmid DNA Sutable for Mammalian Cell Transfections. BioTechniques 23 (3), 424-427].

MT-4 cells were cultured as described above for 3 days to a concentration of 1.6 million / ml, washed twice with RPMI medium, suspended in the desired volume of medium to a concentration of 1 × 10 ⁷ cells / ml. 40 μg of plasmid DNA was added to 800 μl of the cell suspension, incubated for 15 minutes in ice and transferred to an electroporation cuvette. Electroporation is carried out at 400 W, 960 μF (Gene Pulser ^R Transfection Apparatus, Bio-Rad). The pulse duration is 31-36 ms. After electroporation, cells are transferred to RPMI medium supplemented with 10% fetal bovine serum. The efficiency of introducing plasmid DNA is 92-98% (assessed by recording the appearance of green fluorescence using an Olimpus fluorescence microscope). Cells are infected 3 days after electroporation with various multiplicity of infection. For infection, use the supernatant of cells infected with the GKV-4046 strain of HIV-1 with a multiplicity of infection of 2-5 × 10 ^-1 , 2-5 × 10 ^-2 and 2-5 × 10 ^-3 infectious units per cell. The plate is incubated at 37 ° C for four days in an atmosphere of 5% CO ₂ . After 72 hours, samples were taken to quantify the virus-specific protein p24 by direct enzyme immunoassay. Concentration and cell viability are evaluated by trypan blue exclusion. The effect of introducing genetic constructs into cells on virus reproduction is assessed by the accumulation of the virus-specific p24 antigen. The results are shown in table 3.

Table 3 No. Design name % suppression of virus production one anti-HIV-2 83 2 anti-HIV-3 67 3 anti-HIV-4 88 four anti-HIV-5 69 5 anti-gag-1 85

In cells containing genetic constructs, a significant suppression of HIV-1 reproduction is observed compared to untransfected cells (up to 88%). A pronounced antiviral effect is observed upon infection of cells with a high concentration of the virus (the number of viral particles is equivalent to 1081.5 ng p24). The maximum suppression of the virus was detected upon infection of cells 72 hours after the introduction of DNA genetic constructs.

Claims (12)

1. The anti-HIV-1 genetic construct based on the siSTRIKE-neo vector producing siRNA - an inhibitor of the reproduction of the human immunodeficiency virus type 1, containing a 23 bp HIV-1 genome fragment corresponding to the conserved region of the reverse transcriptase domain of the HIV-1 genome (2435 ... 2457):
5'AAACATCAGAAAGAACCTCCATTcttcctgtcaAATGGAGGTTCTTTCTGATGTTTtttttt3 ',
where the lowercase letters indicate the region of the loop between the parts of the palindrome capable of forming a double-stranded duplex, as well as the region (T) ₅ at the 3 'end, the region corresponding to the sense strand of the virus RNA is shown in bold in the line on the left and the antisense in the right. 2. The anti-HIV-2 genetic construct based on the siSTRIKE-neo vector producing siRNA - an inhibitor of the reproduction of human immunodeficiency virus type 1, containing a fragment of 19 bp length corresponding to the conserved region of the reverse transcriptase domain in the HIV-1 genome (2309 ... 2327):
5'ATAGTGATCTACCAATACActtcctgtcaTGTATTGGTAGATCACTATttttt3 ',
where the lowercase letters indicate the region of the loop between the parts of the palindrome capable of forming a double-stranded duplex, as well as the region (T) ₅ at the 3 'end, the region corresponding to the sense strand of the virus RNA is shown in bold in the line on the left and the antisense in the right. 3. The genetic construction of anti-HIV-3 based on the siSTRIKE-neo vector producing siRNA - an inhibitor of the reproduction of human immunodeficiency virus type 1, containing a fragment of 19 bp length corresponding to the conserved region of the integrase domain in the HIV-1 genome (3905 ... 3923):
5'GTAGTGGAATCTATGAATActtcctgtcaTATTCATAGATTCCACTACttttt 3 ',
where the lowercase letters indicate the region of the loop between the parts of the palindrome capable of forming a double-stranded duplex, as well as the region (T) ₅ at the 3 'end, the region corresponding to the sense strand of the virus RNA is shown in bold in the line on the left and the antisense in the right. 4. The anti-HIV-4 genetic construct based on the siSTRIKE-neo vector producing siPHK, an inhibitor of the reproduction of human immunodeficiency virus type 1, containing a fragment of 19 bp length corresponding to the conserved region of the U protein domain in the HIV-1 genome (5366 ... 5384):
5'GGACCATAGTGTATATAGActtcctgtcaTCTATATACACTATGGTCCttttt 3 ',
where the lowercase letters indicate the region of the loop between the parts of the palindrome capable of forming a double-stranded duplex, as well as the region (T) ₅ at the 3 'end, the region corresponding to the sense strand of the virus RNA is shown in bold in the line on the left and the antisense in the right. 5. The anti-HIV-5 genetic construct based on the siSTRIKE-neo vector producing siPHK, an inhibitor of the reproduction of human immunodeficiency virus type 1, containing a fragment of 18 bp length corresponding to the conserved region of the GP120 protein domain in the HIV-1 genome (6380 ... 6397):
5'GGACAAGCCTTCTATACActtcctgtcaTGTATAGAAGGCTTGTCCttttt 3 ',
where the lowercase letters indicate the region of the loop between the parts of the palindrome capable of forming a double-stranded duplex, as well as the region (T) ₅ at the 3 'end, the region corresponding to the sense strand of the virus RNA is shown in bold in the line on the left and the antisense in the right. 6. Genetic construct anti-GAG-1 based on the siSTRIKE-neo vector producing siRNA - an inhibitor of the reproduction of human immunodeficiency virus type 1, containing a fragment of 19 bp length corresponding to the conserved region of the P17 protein domain in the HIV-1 genome (3 ... 21):
5'GCGAGAGCGTCAATATTAAttcaagagaTTAATATTGACGCTCTCGCttttt 3 ',
where the lowercase letters indicate the region of the loop between the parts of the palindrome capable of forming a double-stranded duplex, as well as the region (T) ₅ at the 3 'end, the region corresponding to the sense strand of the virus RNA is shown in bold in the line on the left and the antisense in the right. 7. The anti-HIV-1-27 genetic construct based on the siSTRIKE-neo vector producing siPHK, an inhibitor of the reproduction of the human immunodeficiency virus type 1, containing a 27 bp fragment corresponding to the conserved region of the reverse transcriptase domain in the HIV-1 genome (2435 ... 2461 ):
5'AAACATCAGAAAGAACCTCCATTCCTTttcaagagaAAGGAATGGAGGTTCTTTCTGATGTTTTtttt 3 ',
where the lowercase letters indicate the region of the loop between the parts of the palindrome capable of forming a double-stranded duplex, as well as the region (T) ₅ at the 3 'end, the region corresponding to the sense strand of the virus RNA is shown in bold in the line on the left and the antisense in the right. 8. The anti-HIV-2-27 genetic construct based on the siSTRIKE-neo vector producing siRNA - an inhibitor of the reproduction of the human immunodeficiency virus type 1, containing a 27 bp fragment corresponding to the conserved region of the reverse transcriptase domain in the HIV-1 genome (2304 ... 2330 ):
5'CAGAAATAGTGATCTACCAATACATGGttcaagagaCCATGTATTGGTAGATCACTATTTCTGttttt 3 ',
where the lowercase letters indicate the region of the loop between the parts of the palindrome capable of forming a double-stranded duplex, as well as the region (T) ₅ at the 3 'end, the region corresponding to the sense strand of the virus RNA is shown in bold in the line on the left and the antisense in the right. 9. The anti-HIV-3-27 genetic construct based on the siSTRIKE-neo vector producing siRNA, an inhibitor of the reproduction of the human immunodeficiency virus type 1, containing a 27 bp fragment corresponding to the conserved region of the integrase domain in the HIV-1 genome (3901 ... 3927) :
5'AGGAGTAGTGGAATCTATGAATAAAGAttcaagagaTCTTTATTCATAGATTCCACTACTCCTttttt 3 ',
where the lowercase letters indicate the region of the loop between the parts of the palindrome capable of forming a double-stranded duplex, as well as the region (T) ₅ at the 3 'end, the region corresponding to the sense strand of the virus RNA is shown in bold in the line on the left and the antisense in the right. 10. The anti-HIV-4-27 genetic construct based on the siSTRIKE-neo vector producing siRNA - an inhibitor of the reproduction of the human immunodeficiency virus type 1, containing a 27 bp fragment corresponding to the conserved region of the U protein domain in the HIV-1 genome (5361 .. 5388):
5'AGTGTGGACCATAGTGTATATAGAATATtcaagagATATTCTATATACACTATGGTCCACACTttttt 3 ',
where the lowercase letters indicate the region of the loop between the parts of the palindrome capable of forming a double-stranded duplex, as well as the region (T) ₅ at the 3 'end, the region corresponding to the sense strand of the virus RNA is shown in bold in the line on the left and the antisense in the right. 11. The anti-HIV-5-27 genetic construct based on the siSTRIKE-neo vector producing siRNA, an inhibitor of the reproduction of human immunodeficiency virus type 1, containing a 27 bp fragment corresponding to the conserved region of the GP120 protein domain in the HIV-1 genome (6376 ... 6402 ):
5'ACCAGGACAAGCCTTCTATACAAACAGttcaagagaCTGTTTGTATAGAAGGCTTGTCCTGGTttttt 3 ',
where the lowercase letters indicate the region of the loop between the parts of the palindrome capable of forming a double-stranded duplex, as well as the region (T) ₅ at the 3 'end, the region corresponding to the sense strand of the virus RNA is shown in bold in the line on the left and the antisense in the right. 12. The anti-GAG-1-27 genetic construct based on the siSTRIKE-neo vector producing siRNA, an inhibitor of the reproduction of human immunodeficiency virus type 1, containing a 27 bp fragment corresponding to the conserved region of the P17 protein domain in the HIV-1 genome (1 ... 27 ):
5'GTGCGAGAGCGTCAATATTAAGCGGGGttcaagagaCCCCGCTTAATATTGACGCTCTCGCACttttt 3 ',
where the lowercase letters indicate the region of the loop between the parts of the palindrome capable of forming a double-stranded duplex, as well as the region (T) ₅ at the 3 'end, the regions corresponding to the sense strand of the RNA of the virus are shown in bold in the line on the left and the antisense in the right.

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