RU2748998C1 - Kit for determining ccr5delta32 mutations in human genome - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, specifically to means for determining the CCR5delta32 mutation in the human genome, and can be used to determine and count the number of mutant CCR5delta32 alleles in the studied genetic material during genetic screening and to determine the efficiency of genome editing when using innovative methods of treating HIV infections. A kit has been proposed for determining the CCR5delta32 mutation in the human genome during digital drop multiplex PCR in real time, including forward and reverse primers, a CCR5-78-MT-FAM fluorescent probe with a fluorescent dye molecule 5 (6) -carboxyfluorescein (FAM) covalently stitched to the 5'-end of the sequence and a fluorescence quencher molecule BHQ1 covalently stitched to the 3'-end of the sequence and a CCR5-78-WT-R6G fluorescent probe with covalently 5'-the end of the sequence with a molecule of a fluorescent dye Rhodamine 6G (R6G) and a molecule of a fluorescence quencher BHQ2 covalently stitched to the 3'-end of the sequence.

EFFECT: high-precision counting of CCR5-Δ32 copy number of mutant alleles with a resolution of up to 0.1% due to the high specificity of the reagents of the proposed set.

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Description

The invention relates to the field of medicine, biotechnology, molecular biology, can be used to determine and count the number of mutant CCR5delta32 alleles in the studied genetic material during genetic screening and to determine the efficiency of genome editing when using innovative methods of treating HIV infections.

Today, one of the biggest unsolved biomedical problems is the human immunodeficiency virus, which causes acquired immune deficiency syndrome (AIDS). According to the United Nations Joint Program on HIV / AIDS (UNAIDS), the total number of people infected with HIV in the world is constantly growing and currently amounts to more than 36 million people, of which more than a million people live in Russia.

The human immunodeficiency virus infects CD4 + cells of the immune system: T-helpers, monocytes, macrophages, dendritic cells, as well as Langerhans cells, microglia cells and others. Antiretroviral drugs, used by patients throughout their life, suppress the multiplication of HIV, preventing the introduction of virions into cells and making it difficult to assemble new viral particles, however, they have a high risk of side effects and high cost (Samji N., Cescon A., Hogg RS, Modur SP, Althoff KN, Buchacz K., Burchell AN, Cohen M., Gebo KA, Gill MJ, Justice A., Kirk G., Klein MB, Korthuis PT, Martin J., Napravnik S., Rourke SB, Sterling TR, Silverberg MJ, Deeks S., Jacobson LP, Bosch RJ, Kitahata MM, Goedert JJ, Moore R., Gange SJ, American N., 2013. Closing the Gap: Increases in Life Expectancy among Treated HIV-Positive Individuals in the United States and Canada // PLoS One V. 8. P. 6-13).

In 1998, researchers found that in some cases, exposure to HIV did not lead to cell infection. It turned out that some people have a natural resistance to HIV infection associated with a mutation of the main co-receptor of the virus - CCR5 (Zagury D., Lachgar A., Chams V., Fall LS, Bernard J., Zagury J.-F., Bizzini B., Gringeri A., Santagostino E., Rappaport J., Feldman M., O'Brien SJ, Burny A., Gallo RC, 1998. CC chemokines, pivotal in protection against HIV type 1 infection // PNAS. V 95. P. 3857-3861).

G., Nowak D., Mossner M., Ganepola S.,

A., Alters K., Ph D., Schneider Т., Hofmann J., Kucherer C, Blau O., Blau I.W., Hofmann W.K., Thiel E., Ph D., Hofmann J., Ph D., Kucherer C, Blau O., Blau I.W., Hofmann W.K., Thiel E., 2009. Long-Term Control of HIV by // N. Engl. J. Med. V. 360. P. 692-697).At the moment, a population of people with the CCR5-Δ32 mutation has been described, which consists in the deletion of 32 nucleotides in the sequence of the gene encoding CCR5 and leading to a shift in the reading frame. This mutation in a heterozygous state significantly reduces the risk of HIV infection, in a homozygous state, infection becomes almost impossible (

G., Nowak D., Mossner M., Ganepola S.,

A., Alters K., Ph D., Schneider T., Hofmann J., Kucherer C, Blau O., Blau IW, Hofmann WK, Thiel E., Ph D., Hofmann J., Ph D., Kucherer C , Blau O., Blau IW, Hofmann WK, Thiel E., 2009. Long-Term Control of HIV by // N. Engl. J. Med. V. 360. P. 692-697).

The frequency of the allele with the CCR5-Δ32 mutation can reach up to 14% in Europe, while this mutation has not been detected in Native Africans, Americans and East Asian ethnic groups.

The discovery of natural immunity to HIV prompted scientists to create a new revolutionary method of treating the disease - transplantation of hematopoietic stem cells with a mutation in CCR5. There are at least three precedents of bone marrow transplantation from a donor with CCR5-Δ32 to an immunocompatible patient with HIV infection, which ended in complete recovery of patients (Allers K., Hu G., Loddenkemper S., Rieger K., Thiel E., Schneider T., 2011. Evidence for the cure of HIV infection by CCR5 DELTA 32 / DELTA 32 stem cell transplantation // Blood. V. 117. P. 2791-2799; Gupta RK, Abdul-jawad S., Mccoy LE, Mok HP, Peppa D., Salgado M., Martinez-picado J., Nijhuis M., Wensing AMJ, Lee H., Grant P., Nastouli E., Lambert J., Pace M., Salasc F., Monit C. , Innes AJ, Muir L., Waters L., 2019. HIV-1 remission following CCR5Δ32 / Δ32 haematopoietic stem-cell transplantation // Nature. V. 568. P. 244-248).

However, for obvious reasons, it is very difficult to find such compatible donors for the overwhelming majority of those infected; therefore, there is a request for the creation of ultra-precise high-throughput genetic screening systems for the search for CCR5-Δ32 mutant alleles.

Also, another revolutionary approach can be considered attempts to optimize the CRISPR / Cas9 genome editing system to obtain human cells with artificial CCR5 knockout and the creation of an artificial CCR5-Δ32 mutation.

In a number of works, it was possible to obtain induced stem cells with deletions in the CCR5 gene sequence that demonstrated resistance to HIV infection (Kang X., He W., Huang Y., Yu Q., Chen Y., 2016. Introducing precise genetic modifications into human 3PN embryos by CRISPR / Cas-mediated genome editing // J. Assist. Reprod. Genet; Ye L., Wang J., Beyer AI, Teque F., Cradick TJ, Qi Z., Chang JC, Bao G., Muench MO , Yu J., Levy JA, Kan YW, 2014. Seamless modification of wild-type induced pluripotent stem cells to the natural CCR5Δ32 mutation confers resistance to HIV infection. // Proc. Natl. Acad. Sci. USAV 111. P. 9591 -9596).

This approach can be used for both CCR5 knockout in postnatal hematopoietic stem cells intended for transplantation to HIV patients (Xu L, Yang H, Gao Y, Chen Z, Xie L, Liu Y, Liu Y, Wang X, Li H, Lai W , He Y, Yao A, Ma L, Shao Y, Zhang B, Wang C, Chen H, Deng H. CRISPR / Cas9-Mediated CCR5 Ablation in Human Hematopoietic Stem / Progenitor Cells Confers HIV-1 Resistance In Vivo Mol Ther. 2017 Aug 2; 25 (8): 1782-1789. Doi: 10.1016 / j.ymthe. 2017.04.027. Epub 2017 May 17. PMID: 28527722; PMCID: PMC5542791) and for the targeted creation of CCR5-Δ32 mutations in the genome human embryo (T.A.Kodyleva, A.O. Kirillova, E.A.Tyshchik, V.V. Makarov, A.V. Khromov, V.A.Gushchin, A.N. Abubakirov D.V. Rebrikov, G. T. Sukhikh. Efficiency of creation of CCR5Ddelta32 deletion by CRISPR-Cas9 method in human embryos.Vestnik RSMU; 2018 (4): 80-84; DOI: 10.24075 / vrgmu.2018.052; Kang X., He W., Huang Y., Yu Q., Chen Y., 2016. Introducing precise genetic modifications into human 3PN embryos by CRISP R / Cas-mediated genome editing // J. Assist. Reprod. Genet).

Since the efficiency of editing the human genome using CRISPR / Cas9 systems is still one of the most controversial arguments against the translation of this method into medicine and clinics, the development of methods for searching for CCR-Δ32 mutations and high-precision measurement of the proportion of mutant alleles is still relevant.

At the time of filing the application from open sources, it was known about the following closest analogs:

Patent RU 2563172, C1 describes a method for the specific detection of allelic polymorphism CCR5 delta 32 in a cord blood sample, which can be used for screening of cord blood samples supplied to the cord blood bank. However, the known test system is based on a primitive analysis of PCR results using gel electrophoresis, which does not provide a quantitative assessment of the content of mutant alleles in the analyzed mixture.

Application RU 2003111838, A describes a method for the simultaneous specific detection of allelic polymorphisms CCR5delta32 and CCR5M303, which can also be used for screening examinations. This test system is also based on a primitive analysis of the results of PCR and restriction analysis of amplicons using gel electrophoresis, which also does not provide a quantitative assessment of the content of mutant alleles in the analyzed mixture.

A known method for the simultaneous detection of CCR5delta32 mutations and HLA-B * 5701 allelic polymorphism using multiplex PCR with endpoint analysis, that is, using gel electrophoresis (Rosi A, Meini G, Materazzi A, Vicenti I, Saladini F, Zazzi M. Low-cost simultaneous detection of CCR5-delta32 and HLA-B * 5701 alleles in human immunodeficiency virus type 1 infected patients by selective multiplex endpoint PCR.J Virol Methods. 2015 Nov; 224: 102-4.doi: 10.1016 / j.jviromet .2015.08.020. Epub 2015 Sep 2. PMID: 26341061). This analogue also does not allow for a quantitative assessment of the content of mutant alleles in the analyzed mixture.

L, Hodek J, Machala L,

M, Weber J. Prevalence and the role of CCR5Δ32 heterozygosity in disease progression in HIV positive patients in the Czech Republic. Epidemiol Mikrobiol Imunol. 2019 Winter; 68(3):138-143. English. PMID: 31914779).As a prototype, we have chosen a method for semi-quantitative detection of CCR5delta32 mutation using two primers and two fluorescent probes, one of which is written in the wild-type sequence, and the other is written in the sequence with the CCR5delta32 mutation (

L, Hodek J, Machala L,

M, Weber J. Prevalence and the role of CCR5Δ32 heterozygosity in disease progression in HIV positive patients in the Czech Republic. Epidemiol Mikrobiol Imunol. 2019 Winter; 68 (3): 138-143. English. PMID: 31914779).

In the prototype method, PCR takes place in an amplifier with the function of measuring fluorescence in real time (Real-Time PCR), while in the case of finding a wild-type allele, an amplification curve with FAM fluorescence is detected, in the case of finding a homozygous allele with CCR5delta32 mutation, a mutation is detected amplification fluorescence ROX. If both alleles are found, both curves will be detected in both channels.

This method assumes only conditionally quantitative determination of the copy number of the allele with the CCR5delta32 mutation, since the accuracy of this method is limited by the possibility of separating two groups of amplification curves and is no more than 33%.

The problem to be solved by this invention is the creation of a kit that allows for accurate determination with measurement of the proportion of mutant alleles CCR5-Δ32 mutations in the presented genetic material in relation to the alleles "wild type".

The technical result achieved when using the proposed set is to ensure high-precision counting of CCR5-Δ32 copy number of mutant alleles in the presented genetic material with a resolution of up to 0.1% (detection of 1 copy of a mutant allele per 1000 copies of wild-type alleles, the ability to reliably distinguish the measurement result of one copies of the mutant allele per 1000 copies of the "wild type" allele from two copies of the mutant allele per 1000 copies of the "wild type" allele) due to the high specificity of the reagents of the proposed set.

Original oligonucleotide primers and fluorescent probes have been proposed, which make it possible to distinguish different alleles of the CCR5 gene: mutant CCR5-Δ32 alleles from healthy “wild-type” alleles during digital drop multiplex polymerase chain reaction (PCR).

The essence of the invention is as follows.

The proposed kit for determining the CCR5delta32 mutation in the human genome when conducting digital drop multiplex PCR, comprising a forward primer having the sequence SEQ ID NO: 1; a reverse primer having the sequence of SEQ ID NO: 2; fluorescent probe CCR5-78-MT-FAM, having the sequence SEQ ID NO: 3, with a fluorescent dye molecule 5 (6) -carboxyfluorescein (FAM) covalently linked to the 5 'end of the sequence and a quencher molecule covalently linked to the 3' end of the sequence fluorescence BHQ1; and a CCR5-78-WT-R6G fluorescent probe having the sequence SEQ ID NO: 4, with a Rhodamine 6G (R6G) fluorescent dye molecule covalently linked to the 5 'end of the sequence and a BHQ2 fluorescence quencher molecule covalently linked to the 3' end of the sequence.

Thus, the ability to accurately count the number of copies of the CCR5-Δ32 mutation is achieved by using two primers flanking the locus containing the CCR5-Δ32 deletion:

FW-Primer: 5'-CCCAGGAATCATCTTTACCA-3 '

RV-Primer: 5'-GACACCGAAGCAGAGTTT-3 '

and two fluorescent probes complementary to the amplicon sequence, one of which detects the gene region in which the CCR5-Δ32 mutation occurs, and the second detects the conserved part of the amplicon and serves for normalization:

CCR5-78-MT-FAM: [FAM] -5'-CAGTCAGTATCAATTCTGGAAGA-3 '- [BHQ1]

CCR5-78-WT-R6G: [R6G] -5'-TCATCTGCTACTCGGGAAT-3 '- [BHQ2].

In this case, the forward primer FW-Primer for amplifying a fragment of the CCR5 gene, which may contain the CCR5delta32 mutation, has the sequence SEQ ID NO: 1.

The reverse primer RV-Primer for amplifying the CCR5 gene fragment, which may contain the CCR5delta32 mutation, has the sequence SEQ ID NO: 2.

The CCR5-78-MT-FAM fluorescent probe with FAM fluorescent dye and BHQ1 quencher for detecting a portion of the CCR5 gene that may contain the CCR5delta32 mutation has the sequence SEQ ID NO: 3.

Fluorescent probe CCR5-78-WT-R6G with fluorescent dye R6G and quencher BHQ2 detects a conserved region of the CCR5 gene for normalization and has the sequence SEQ ID NO: 4.

For the design of primers and probes, bioinformatic analysis was carried out, where the sequence of the CCR5 gene from the database of the National Center for Biotechnology Information U.S. was used as the initial information. National Library of Medicine (Gene ID: 1234, updated on 3-Dec-2017). The nucleotide sequence of the Δ32 deletion was taken from the same database.

Further, to implement the invention, it is necessary to carry out a series of PCR reactions using a system (installation) for digital drop PCR, for example, Bio-Rad QX200 Droplet Digital PCR System, using reagents designed for a specific system (master mix containing the necessary components of PCR reactions, such as Taq polymerase, dNTP, salt solutions, etc.). Preparation of a mixture for digital PCR follows the following protocol:

The composition of the mixture per hole:

1.2x ddPCR supermix for probes (Bio-Rad) 11 μl 2. Primer straight FW-Primer 10 μM 2 μl 3. Reverse primer RV-Primer 10 μM 2 μl 4. Fluorescent probe CCR5-78-MT-FAM 10 μM 0.6 μl 5. Fluorescent probe CCR5-78-WT-R6G 10 μM 0.6 μl 6. Water 0.8 μl 7. DNA solution (analyzed genetic material) 5 μl

The total volume should be 22 μl, the final concentration of primers is 900 nM, the final concentration of probes is 250 nM, it is recommended to prepare a DNA solution so that 5 μl contains from 50 ng to 350 ng of DNA. Further, using the device for generating drops, it is necessary to prepare the emulsion of the drops. After the completion of the generation of drops, the plate with drops is sealed with foil for 5 seconds at 180 degrees. The sealed plate fits into any thermal cycler that supports skirted 96-well plates (eg C1000 Thermal Cycler, Bio-Rad). The thermal cycling protocol is presented in the table.

After amplification, the plateau is transferred to the instrument for detecting fluorescence in droplets via the FAM and R6G (HEX) channels. The analysis of the results is carried out using the program supplied with the device - QuantaSoR SoAware Bio-Rad. As a typical result, the program produces two fluorescence plots (FAM and R6G) in 1D format and a combined multiplex plot in 2D format.

FIG. 1 shows an example of the analysis of the result of digital drop PCR in the form of graphs in 1D format; upper graph - measurement of fluorescence in the FAM channel; bottom graph - measurement of fluorescence in the R6G channel; each point on the graphs is an analyzed drop; on the ordinate scale — the fluorescence intensity; on the abscissa scale — the ordinal number of the analyzed drop; lower cluster of drops (colorless) - drops without a matrix; upper clusters - positive drops where PCR took place; in this case, we see the analysis of the "wild type" matrix, since all droplets fluoresce in both channels.

FIG. 2 shows an example of the analysis of the result of digital drop PCR in the form of a 2D plot: on the ordinate scale - the fluorescence intensity in the FAM channel, on the abscissa scale - the fluorescence intensity in the R6G channel; negative cluster of drops (colorless) - bottom left; positive cluster of droplets (orange), where PCR took place - top right; in this case, we see the analysis of the "wild type" matrix, since all droplets fluoresce in both channels.

FIG. 3 shows an example of the analysis of the result of digital drop PCR in the form of graphs in 1D format: upper graph - measurement of fluorescence in the FAM channel; bottom graph - measurement of fluorescence in the R6G channel; each point on the graphs is an analyzed drop; on the ordinate scale — the fluorescence intensity; on the abscissa scale — the serial number of the analyzed drop; lower cluster of drops (colorless) - drops without a matrix; upper clusters - positive drops where PCR took place; in this case, we see an analysis of the matrix containing the homozygous CCR5-Δ32 mutation, since all droplets fluoresce in the R6G channel, but not in the FAM channel.

FIG. 4 shows an example of the analysis of the result of digital drop PCR in the form of a 2D plot; on the ordinate scale - fluorescence intensity in the FAM channel; on the abscissa scale - fluorescence intensity in the R6G channel; negative cluster of drops (colorless) - bottom left; a positive cluster of drops (green), where the PCR took place - at the bottom right; in this case, we see an analysis of the matrix containing the homozygous CCR5-Δ32 mutation, since all droplets fluoresce in the R6G channel, but not in the FAM channel.

Thus, when analyzing the plots in 1D format (Fig. 1), two clusters of drops are seen, the lower one is negative, where the template (DNA) for amplification did not get, and the upper one is positive, where the template (DNA) got, where the PCR successfully passed and a fluorescence signal appears. The program automatically separates both clusters and calculates the values.

When analyzing the graphs in 2D format (Fig. 2), both clusters of cells are clearly distinguishable, and are also automatically separated. If the DNA does not contain a mutation in the analyzed CCR5 locus, we see the same luminescence of all drops in the FAM and R6G channels (examples for "wild type" DNA are shown in Fig. 1 and Fig. 2).

If the DNA contains the CCR5-Δ32 mutation, we see the glow of drops in R6G, but we do not see the glow in the FAM channel (examples for DNA with a homozygous CCR5-Δ32 mutation are shown in Fig. 3 and Fig. 4).

Since the digital drop PCR format quantitatively analyzes the content of the matrix in the initial mixture in pieces, it allows highly accurate measurement of the ratio of mutant alleles compared to the wild type (up to 0.1%).

The basis of the method with the proposed kit for measuring the copy number of CCR5-Δ32 mutant alleles is synthesized short DNA oligonucleotides and an instrumental base for conducting digital drop PCR. The technology for the synthesis of DNA oligonucleotides is quite well mastered by the biochemical industry. Digital drip PCR instruments are widely available and can be purchased or used at Shared Centers.

The description of the present invention is illustrative and does not limit the scope of the claims. For qualified specialists, it is obvious that there are possible variations and modifications of the invention associated with its specific implementation, without going beyond the scope of the claims.

The figures in the application are presented in a form sufficient for the understanding of the principles of the invention by specialists in this field, and in no way limit the scope of the present invention.

Sequence listing

<110> Federal State Autonomous Educational Institution of Higher Education “Russian National Research Medical University named after N.I. Pirogov "of the Ministry of Health of the Russian Federation (Federalnoe gosudarstvennoe autonomnoe obrazovatelnoe uchrezhdenie vysshego obrazovaniya" Rossijskij natsionalnyj issledovatelskij meditsinskij universitet imeni N.I. Pirogova "

<120> Kit for detecting CCR5delta32 mutations in the human genome

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<223> Forward FW-Primer to amplify a CCR5 gene fragment that may contain the CCR5delta32 mutation

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<223> Reverse primer RV-Primer to amplify a fragment of the CCR5 gene that may contain the CCR5delta32 mutation

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<223> CCR5-78-WT-R6G fluorescent probe with R6G fluorescent dye and BHQ2 quencher detects a conserved region of the CCR5 gene for normalization

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Claims (1)

Kit for detecting CCR5delta32 mutations in the human genome during digital drop multiplex polymerase chain reaction (PCR) in real time, comprising a forward primer having the sequence SEQ ID NO: 1, a reverse primer having the sequence SEQ ID NO: 2, a fluorescent probe CCR5 -78-MT-FAM having the sequence SEQ ID NO: 3, with a fluorescent dye molecule 5 (6) -carboxyfluorescein (FAM) covalently stitched to the 5'-end of the sequence and a fluorescence quencher molecule BHQ1 covalently stitched to the 3'-end of the sequence, fluorescent probe CCR5-78-WT-R6G, having the sequence SEQ ID NO: 4, with a molecule of the fluorescent dye Rhodamine 6G (R6G) covalently linked to the 5'-end of the sequence and a molecule of the fluorescence quencher BHQ2 covalently linked to the 3'-end of the sequence.

Images