Cartridge genetic maker expressing three biologically active siphk, effectively attacking human immunodeficiency virus and ccr5 gene transcripts by means of rna-interference

FIELD: medicine.

SUBSTANCE: what is described in a cartridge genetic maker based on GeneClip-Ul-neo vector producing three siRNA - reproduction inhibitors of human immunodeficiency virus type 1 and human CCR5 gene. The maker contains a fragment of the length 27 bp related to a conservative region of a HIV-1 genome reverse transcriptase domain, a fragment of the length 27 bp related to the other conservative region of the HIV-1 genome reverse transcriptase domain, as well as a fragment 19 bp related to CCR5 gene mRNA. The invention can be used in medicine and scientific research.

EFFECT: invention allows producing more effective anti-HIV preparations based on interfering RNA produced in cells by means of the introduced cartridge genetic maker containing palindromes for three siRNA production.

3 dwg, 2 tbl, 2 ex


The invention relates to the fields of medical and molecular genetics, and gene therapy, and is associated with the previously identified targets for RNA interference in human immunodeficiency virus type 1 (HIV-1) to create a genetic cassette design. This cassette expresses two biologically active siPHK that leads to the attack of the relevant targets in the transcripts of the virus, and potent inhibition of its reproduction in human cells. The specified cassette genetic design Express hairpin RNAS that are processed in vivo to the biologically active siPHK. Last effectively delivered in protein complexes responsible for cleavage of transcripts of the virus, and this is achieved by the inhibition of its reproduction. In addition, the cassette expresses and new biologically active siPHK attacking mRNA gene CCR5 person. Specified gene encodes a co-receptor, facilitating penetration of the virus in T-lymphocytes of human rights. This genetic construct can be used in medicine for gene therapy of AIDS and HIV infection, as well as in scientific research for powerful anti-HIV products.

Currently, the main approach for the treatment of AIDS and HIV infection is a prescription antiviral chemotherapy, including drugs that act on key fermentive-1 - reverse transcriptase, integrase, and protease. However, despite the large number of drugs, there is the issue of the effectiveness of antiviral therapy. The main reason for treatment failure - adaptive mutagenesis of the virus, leading to the emergence of variants of viruses resistant to antiviral drugs. Serious problems are also toxicity and high cost of used medicines.

It is known the 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 Century, 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., S. Escaich, 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 multiple HIV genes, which reduces its activity.

The closest to this invention are genetic constructs that give expression siPHK within HIV-infected cells (RF Patent 2324738, "Genetic design based on the vector plasmid pEGFP-Nl, producing siPHK inhibitors reproduction of human immunodeficiency virus type 1 (options)".

The present image is the buy differs in the present genetic structure expressed not one but three siPHK, two of which focused on two other targets in the transcripts of the virus, and one targeted to the mRNA of the gene CCR5 human encoding a cellular co-receptor involved in the penetration of the virus in T-lymphocytes. In addition, in the present genetic structure uses a different initial vector.

The technical result of the invention is the use of two previously identified targets RNA intreference in the virus (patent of the Russian Federation dated 29.08.2008, registration number 2008134932) and one new targets CCR5 mRNA described in this Application, to obtain a higher effect of suppressing the virus. This cassette genetic structure contains three palindrome intended for the education of RNA hairpins". The expression of such constructs in human cells leads to the formation of three interfering RNA (siPHK). The proposed genetic structure encode biologically active siPHK, which were selected using non-viral test systems using cultures of human cells.

Presents the technical result is achieved by creating a genetic cassette design on the basis of the vector GeneClip™ U1 Neomycin (Ac. number AY745746), .pdf.

Created genetic structure length 195 bp contain stawk the DNA specified in Table 1:

Table 1 shows 19-27-nucleotide sequence corresponding to the transcripts of the virus or CCR5 mRNA. Lowercase letters in bold in the following order specified regions corresponding to the semantic chain mRNA virus (2435..2461), antisense chain; semantic chain mRNA virus (2304..2330), antisense chain; semantic chain mRNA gene CCR5 (47..65), antisense chain. The DNA sequence presented in orientation 5'-3'. Lowercase letters shown texts loops and spacers containing artificial sites for restricted EcoRI or BamHI (bold lowercase letters). Loops are formed in the transcript after annealing of complementary areas of palindromes. The texts of genetic constructs correspond to the strain of HIV-1 subtype A (HIV-1 subtype A), the number of sequences in GenBank - AF316544. The numbering of the CCR5 gene region is specified by a sequence of GenBank - U54994. This design is intended for defeat of two regions of mRNA that encode conservative region of the reverse transcriptase (RT) virus, and the 5' region of the mRNA of the gene CCR5 person in the first codons.

Specified genetic structure was tested using non-viral systems. This system is intended for quantitative evaluation of biological activity siPHK, which are formed in vivo by expression of genetices the x structures. It was created on the basis of the vector psiCHECK™-2 (Promega, number sequences in GenBank AY535007). The vector contains a gene of the Firefly luciferase gene and the luciferase coral (Renilla). These luciferase cause the illumination of different wavelengths and their expression can be measured using a luminometer. 3' end non-coding region of the gene Renilla inserted a short region of the viral genome (domains)corresponding to the selected target RNA interference in the transcripts of the virus. The domains were obtained using chemical synthesis of DNA oligonucleotides. Table 2 presents the texts of the domains that cloned in the vector psiCHECK™-2 sites restricts XhoI and NotI.

In table 2 lowercase letters shows the region containing the sites of the restriction endonucleases XhoI and NotI, and two spacer between fragments of the genome of HIV-1 and CCR5 cDNA. Bold font shows the region of the viral genome and cDNA CCR5 length of 28 bp each, which correspond to the target RNA interference. The numbering of the areas are listed according to the sequence of the virus (AF316544) and CCR5 cDNA (U54994) in the following order: a target for anti-HIV-1, anti-HIV-2, anti-CCR5-8.

Testing the biological activity of the cassette is carried out in experiments on co-transfection culture of human cells. Using two DNA preparation: cassette genetic constructs containing the promoter U1 RNA polymerase I coding coding three siPHK, as well as a DNA plasmid-based vector psi-CHECK-2, containing three cloned domain relevant data siPHK (target RNAi) (table 2). In human cells at the genetic structures is expressed paste containing palindromes. Synthesized RNA forms a hairpin, which is a natural substrate of Disera, enzyme, the clip 21 nucleotide duplexes RNA from studs. If Dicer working on the duplex RNA longer than 19 base pairs (natural substrate), then he, being associated with double-stranded siPHK, actively participate in its "loading" in the protein complex RISC. In this complex there is an "unwinding" of the circuits siPHK and freeing it from one of them (the so-called "passenger"-chain). If the complex remains antisense siPHK, it is used as a "gunner", recognizing the complementary target transcripts cells. After binding of the complex with the corresponding mRNA is cutting one of its proteins, which leads to shut down expression of the corresponding gene of the virus and violates its reproduction in the cell host. In transfected cells Renilla mRNA containing a 3'-non-coding region of the test target RNA interference, is cut, which leads to a sharp decrease in the blue glow, caused by the luciferase (478 nm). Yellow-green is e glow, called luciferase Firefly Photinus pyralis (557 nm), encoded by the same DNA psiCHECK-2, serves as an internal control.

This study used ten criteria for selecting the sequence of the target RNA interference of mRNA of a gene CCR5. They are important to ensure that the antisense chain siPHK remained in the RISC complex. Each are given in Table 1 inserts in the genetic structures corresponds to at least ten of the eleven following criteria were used to select crustal 19-sequence polymorphisms siPHK:

composition G/C 30-52%

- at least 3 A/U in position 15-19 semantic chain

- lack of internal repeats

- And at position 19 of the sense circuit

And in position 3, the semantic chain

- U at position 10 of the sense circuit

- no G/C at position 19 of the sense circuit

- the absence of G at position 13 of the sense circuit

- the absence of homopolymer tracts (A)4-5or (T)4-5in the semantic chain

- localization of a target in a conservative region of the viral genome

- lack of homology to known transcripts in the human genome.

Part of the above selection criteria biologically active siPHK 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 presented on the website

See the table 1 genetic cassette design to deliver a targeted effect on the mRNA of the virus in human cells using siPHK, containing substances that occur in cells and CCR5 mRNA. therapeutic result of this effect is the suppression of the reproduction of HIV-1 in human cells.

For human immunodeficiency virus the first type is characterized by a rapid emergence and selection of mutant variants with resistance to various anti-viral drugs. To solve viral variability obtained genetic cassette design, attacking different targets and therefore largely independent of the variability of the virus.

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

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

Figure 3 presents the results of examining the effectiveness of RNA interference caused created genetic cassette design on non-viral test the system.

Example 1. Description of the genetic structure and method of reception

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

the start site of RNA polymerase T7: 1

U1 premotor RNA polymerase II person: 46-438

10 bp spacer: 439-448

U1 terminator: 449-465

the promoter of RNA polymerase SP6: 527-546

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

signal the beginning of replication of SV40 virus: 1114-1179

gene neo: 1251-2045

synthetic poly(A): 20802128

the gene of β-lactamase: 3080-3940

the promoter of RNA polymerase T7: 4742-3.

In this vector, which comes Promega in linear form, insert a chemically synthesized DNA oligonucleotides, which correspond to the genetic structures that produce hairpin RNA (table 1).

Vector psiCHECK-2 is registered in GenBank (Accession # AY535007), has a size 6,27 kb and is characterized in that it contains:

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

chimeric intron: 489-621

the promoter of RNA polymerase T7: 666-684

gene luciferase Relilla: 694-1629

polylinker: 1636-1680

synthetic poly(A): 1688-1736

the promoter HSK-TK: 1744-2496

gene luciferase Firefly: 2532-4184

late signal poly(A) SV40: 4219-4440

the gene of β-lactamase: 4587-5447.

In this vector in the sites XhoI and NotI of polylinker insert chemically synthesized DNA oligonucleotides that correspond to the selected target RNA interference (table 2).

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

On the eve of the co-transfection (18-20 hours) cells NEC scatter in the wells of a 24-hole tablet Nunc 50 testlets in 500 μl of culture medium DMEM (Paneco)containing glutamine and serum (complete medium), per well. On the day of the experiment, prepare DNA samples for co-transfection at room temperature following the way (one experimental point). 200 μl of DMEM medium containing no serum (SFM), add 100 ng DNA genetic structure in the vector GeneClip-U1-neo (see Table 1) and 10 ng DNA plasmids containing the corresponding cloned domain vector psiCHECK-2 (see Table 2). After mixing, add 1 ál viersprong reagent TransFast (Promega), shake the mixture and incubated for her 15 minutes Then sucked off the medium from the wells with those cells, shake the tube with a mixture of DNA TransFast and immediately add the mixture to the wells with cells. After incubation for 1 hour at 37°C in CO2-incubator added to the wells in 600 μl of medium with serum, mix and cuberoot tablet 48-72 hours.

To measure the expression of luciferase medium over the cells in the wells sucked off, add 100 ál of a solution of trypsin-EDTA (Paneco) per well, after 10 min incubation at 37°C in CO2-incubator add 100 ál of complete medium, carry a fully-cell suspension in a test tube, Eppendorf 0.5 ml, precipitated by centrifugation at room temperature in an Eppendorf centrifuge (5 min 2000 rpm), the supernatant is sucked off, suspended cells in 70 μl of 1xPBS and conduct the measurement light luciferase Firefly and Renilla according to the guidelines set Dual-Luciferase Reporter Assay System (Promega) on a luminometer Reporter Microplate Luminometer (Turner BioSystems). Figure 3 shows the results of testing of genetic constructs on neverenoughtime. It is seen that the genetic constructs specifically inhibit expression of the target for 60-96%.

Cassette genetic design of anti-HIV-1-anti-HIV-2-anti-CCR5-8-based vector GeneClip-U1-neo containing the insert in the area of single-stranded ends, indicated in figure 1, producing three siPHK inhibitors reproduction of human immunodeficiency virus type 1 and gene expression of human CCR5 and contain two fragments of the genome of HIV-1 long 27 bp each, corresponding to two conservative areas of the domain of the reverse transcriptase of the genome of HIV-1 (region 2435 virus..2461 shown in the first line, and the region 2304..2330 - second line), and 19 bp region corresponding to the gene CCR5 (47..65, shown in the third line):
5'AAACATCAGAAAGAACCTCCATTCCTTttcaagagaaaggaatggaggttctttctgatgtttgcgaattctgcagaaatagtgatctaccaatacatggactgtccttcccatgtattggtagatcactatttctgatggatcctcggaacaagatggattatcacttcctgtcatgataatccatcttgttcc 3',
where lower case letters shows the area of the loops between the parts of palindromes that can form double-stranded duplexes.


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