Humanized Mouse Model of HIV Infection.

The development of new drugs for the treatment of HIV infection requires testing of their efficacy in a relevant animal model, such as humanized mice, which, unfortunately, are not yet available in Russia. In the present study, we have developed conditions for the humanization of immunodeficient NSG mice with human hematopoietic stem cells. Humanized animals generated during the study showed a high degree of chimerism and harbored repopulation of the entire range of human lymphocytes required for HIV replication in the blood and organs. Inoculation of these mice with HIV-1 virus led to stable viremia, which was confirmed by the presence of viral RNA in blood plasma throughout the entire period of observation and proviral DNA in the organs of animals 4 weeks after HIV infection.

Generation of SARS-CoV-2 Mouse Model by Transient Expression of the Human ACE2 Gene Mediated by Intranasal Administration of AAV-hACE2.

One of the most important steps in the development of drugs and vaccines against a new coronavirus infection is their testing on a relevant animal model. The laboratory mouse, with well-studied immunology, is the preferred mammalian model in experimental medicine. However, mice are not susceptible to infection with SARS-CoV-2 due to the lack of human angiotensin-converting enzyme (hACE2), which is the cell receptor of SARS-CoV-2 and necessary for the entry of the virus into the cell. In present work, it was shown that intranasal administration of the adeno-associated vectors AAV9 and AAV-DJ encoding the hACE2 provided a high level of expression of ACE2 gene in the lungs of mice. In contrast, the introduction of the AAV6 vector led to a low level ACE2 expression. Infection with SARS-CoV-2 of mice expressing hACE2 in the lungs led to virus replication and development of bronchopneumonia on the 7th day after infection. Thus, a simple method for delivering the human ACE2 gene to mouse lungs by intranasal administration of the AAV vector has been proposed. This approach enabled rapid generation of mouse model for studying coronavirus infection.

[Generation of SARS-CoV-2 Mouse Model by Transient Expression of the Human ACE2 Gene Mediated by Intranasal Administration of AAV-hACE2].

One of the most important steps in the development of drugs and vaccines against a new coronavirus infection is their testing on a relevant animal model. The laboratory mouse, with well-studied immunology, is the preferred mammalian model in experimental medicine. However, mice are not susceptible to infection with SARS-CoV-2 due to the lack of human angiotensin-converting enzyme (hACE2), which is the cell receptor of SARS-CoV-2 and necessary for the entry of the virus into the cell. In present work, it was shown that intranasal administration of the adeno-associated vectors AAV9 and AAV-DJ encoding the hACE2 provided a high level of expression of ACE2 gene in the lungs of mice. In contrast, the introduction of the AAV6 vector led to a low level ACE2 expression. Infection with SARS-CoV-2 of mice expressing hACE2 in the lungs led to virus replication and development of bronchopneumonia on the 7th day after infection. Thus, a simple method for delivering the human ACE2 gene to mouse lungs by intranasal administration of the AAV vector has been proposed. This approach enabled rapid generation of mouse model for studying coronavirus infection.

[HIV Restriction Factor APOBEC3G and Prospects for Its Use in Gene Therapy for HIV].

The mechanisms for the protection of the human body from viral or bacterial agents are extremely diverse. In one such mechanism, an important role belongs to the cytidine deaminase APOBEC3 family, which is the factor of congenital immunity and protects the organism from numerous viral agents. One of the proteins of this family, APOBEC3G, is able to protect against Human Immunodeficiency Virus type 1 in the absence of viral protein Vif. In turn, Vif opposes APOBEC3G action, causing polyubiquity of the protein and degradation in the proteasome. The review describes possible ways to increase the anti-HIV activity of APOBEC3G, giving it resistance to viral protein Vif, as well as potential approaches to the use of modified APOBEC3G in gene therapy for HIV.

Immunogenic epitope prediction to create a universal influenza vaccine.

Influenza virus is one of the most rapidly evolving human pathogens and causes significant morbidity and mortality worldwide. This feature enables the virus to avoid natural or vaccine-induced immunity. For this reason, there is an intensive search for new approaches to create a universal influenza vaccine. Here, we propose pipelines based on modern prediction algorithms that allowed us to select 10 B-cell epitopes, 10 CD8+ T-cell epitopes and 6 CD4+ T-cell epitopes from influenza viruses that were characterized by high conservation and antigenicity. These epitopes could be used to create universal vaccines against influenza viruses. In addition, the scripts used in these pipelines are universal and can be used to select epitopes from other pathogens.

[The Titer of the Lentiviral Vector Encoding Chimeric TRIM5α-HRH Gene is Reduced Due to Expression of TRIM5α-HRH in Producer Cells and the Negative Effect of Efla Promoter].

The chimeric protein TRIM5α-HRH is a promising antiviral factor for HIV-1 gene therapy. This protein is able to protect cells from HIV-1 by blocking the virus in the cytoplasm. We are developing protocol of HIV-1 gene therapy, which involves the delivery of the TRIM5α-HRH gene into CD4^(+) T-lymphocytes by lentiviral vectors (LVs). However, LVs containing TRIM5α-HRH have a low infectious titer, which prevents effective T cell modification. Here, we found that the expression of TRIM5α-HRH during pseudoviral particle production in HEK293 T cells, as well as the presence of the Eflα promoter in our construction are responsible for titer reduction. These results allow us to determine the directions for further optimization of LV with the TRIM5α-HRH gene to improve its infectious titer.

Application of real-time PCR to significantly reduce the time to obtain recombinant MVA virus.

Obtaining a pure recombinant Modified Vaccinia Ankara (MVA) virus is a multistage, time-consuming procedure. We describe a novel single-tube real-time PCR which enables determination of the amount of wild type and recombinant viruses and their ratio in plaques. Use of the real-time PCR significantly reduce the time and efforts needed to obtain purified recombinant MVA. The new approach has been applied to generate recombinant MVAs encoding different SARS-COV-2 antigens.

[Retrovirus Restriction Factor TRIM5α: The Mechanism of Action and Prospects for Use in Gene Therapy of HIV Infection].

It is commonly known that the antiviral activity of the TRIM5α protein, the intracellular retrovirus restriction factor, underlies the resistance of the Old World monkeys to HIV-1. This fact suggests that TRIM5α can potentially be used to cure HIV-1 infection in humans. The present review considers the mechanisms of HIV-1 replication inhibition by the TRIM5a protein and the prospects for using it in gene therapy of HIV infection.

[Protection of Lymphocytes Against HIV using Lentivirus Vector Carrying a Combination of TRIM5α-HRH Genes and microRNA Against CCR5].

Gene therapy is considered a promising approach to treating infections caused by human immunodeficiency virus (HIV). One strategy is to introduce antiviral genes into cells in order to impart resistance to HIV. In this work, the antiviral activity of new anti-HIV lentiviral vector pT has been studied. The vector carries a combination that consists of two identical artificial miRNA mic13lg and the TRIM5α-HRH gene. Two mic13lg microRNAs suppress the expression of the CCR5 gene, which encodes the HIV coreceptor and, thus, prevents the penetration of R5-tropic HIV strains into the cell. It has been shown that pT effectively inhibits the expression of CCR5 in both the HT1080 CCR5-EGFP model cell line and in human primary lymphocytes. The second line of protection against R5- and X4-tropic HIV is provided by the TRIM5α-HRH protein, which binds virus capsids after the virus enters the cell. Indeed, when infecting cells of the SupT1 line, which contains four copies of the vector per cell, with the X-4 tropic HIV, more than 1000-fold suppression of viral replication has been observed. The process of generation of the pT vector and conditions of transduction of CD4^(+) lymphocytes were optimized for testing the antiviral activity of the vector on primary human lymphocytes. As a result, the transduction efficiency for the pT vector was 28%. After infection with the R5-tropic strain of the virus, the survival of cells in the culture of lymphocytes with the vector was significantly higher than in the control. However, the complete suppression of HIV replication was not achieved, presumably due to the inadequate fraction of cells that carry the vector in culture. In the future, it is planned to find the best way to enrich the lymphocyte culture with modified cells to increase resistance to HIV.

[Tale nucleases--new tool for genome editing].

The ability to introduce targeted changes in the genome of living cells or entire organisms enables researchers to meet the challenges of basic life sciences, biotechnology and medicine. Knockdown of target genes in the zygotes gives the opportunity to investigate the functions of these genes in different organisms. Replacement of single nucleotide in the DNA sequence allows to correct mutations in genes and thus to cure hereditary diseases. Adding transgene to specific genomic.loci can be used in biotechnology for generation of organisms with certain properties or cell lines for biopharmaceutical production. Such manipulations of gene sequences in their natural chromosomal context became possible after the emergence of the technology called "genome editing". This technology is based on the induction of a double-strand break in a specific genomic target DNA using endonucleases that recognize the unique sequences in the genome and on subsequent recovery of DNA integrity through the use of cellular repair mechanisms. A necessary tool for the genome editing is a custom-designed endonuclease which is able to recognize selected sequences. The emergence of a new type of programmable endonucleases, which were constructed on the basis of bacterial proteins--TAL-effectors (Transcription activators like effector), has become an important stage in the development of technology and promoted wide spread of the genome editing. This article reviews the history of the discovery of TAL effectors and creation of TALE nucleases, and describes their advantages over zinc finger endonucleases that appeared earlier. A large section is devoted to description of genetic modifications that can be performed using the genome editing.

[Downregulation of human CCR5 receptor gene expression using artificial microRNAs].

Chemokine receptor CCR5 is essential for human immunodeficiency virus (HIV) entry into the sensitive cells. The CCR5 inactivation is believed to be one of the promising approaches in HIV therapy, including gene therapy. A powerful mechanism that enables to regulate gene expression is RNA interference which could be exploited to knockdown CCR5 gene. Here, three artificial microRNAs directed against the human CCR5 receptor gene were generated and their silencing activity in indicator cells developed on the basis of the HT1080 cell line was evaluated. Multiplexing of two or more artificial microRNAs in one transcript has been demonstrated to enhance the gene silencing. A 95% reduction of the CCR5 expression has been achieved using the most efficient microRNA combination.

[Safety of retroviral vectors in gene therapy].

Retroviral vectors are widely used in gene therapy and found to be an effective tool for the delivery of genetic constructs into cells. A unique feature of these vectors is the ability to incorporate therapeutic genes into a chromosome that ensures its passage to all progeny cells and enables to cure the diseases requiring genetic correction of dividing cells such as hematopoietic cells or skin cells. Retroviral vectors have been successfully used in gene therapy clinical trials for the treatment of 2 forms of severe combined immunodeficiencies and some other hereditary blood disorders. However, the integration of the vector into the chromosome was accompanied by genotoxicity and caused development of hematologic malignancies in several patients. Later it was shown that genotoxicity is not a general feature of retroviral vectors but it depends on many factors. In the present article we discuss safety issues concerning the use of different retroviral vectors in gene therapy. The description of modern vectors which designed to avoid the genotoxicity and other possible side effects are given.

[Treatment of HIV-infection by means of gene therapy].

Current methods of HIV treatment can contain a progression of the disease; however they do not lead to a cure. Lifelong antiretroviral therapy is therefore necessary, leading to problems of cost and toxicity of chemical drugs. The recent advances in science have allowed a new approach to the HIV-treatment - gene therapy. In the present publication we focus on one strategy of the gene therapy called "intracellular immunization". The strategy is based on the introducing of antiviral genes into the HIV-sensitive cells. We highlight the mechanisms of action of various antiviral genetic agents and discuss some issues concerning target cells and genes delivery. Finally we summarize the results of certain gene therapy clinical trials.

[Progress in gene therapy].

Recent progress in gene therapy, current status of investigations in this area of experimental medicine are reviewed. Much attention is given to gene-therapeutic approaches the efficacy of which is proved in clinical trials.

[Nucleotide sequences of long terminal repeats of the human endogenous retrovirus (LTR HERV-K) on the short arm of chromosome 7: identification, analysis and evaluation of transcriptional activity]. / Posledovatel'nosti dlinnykh kontsevykh povtorov éndogennogo retrovirusa (LTR HERV-K) korotkogo plecha khromosomy 7 cheloveka: poisk, analiz i opredelenie transkriptsionnoi aktivnosti.

Six clones containing long terminal repeat (LTR) sequences of human endogenous retrovirus of the HERV-K family were found in the YAC library (1200 kb) of the short arm of human chromosome 7. The sequence sizes of the three clones corresponded to the full-length LTR (969 bp). The LTR localization was determined using FISH and verified by comparison with the GenBank database. All three DNA fragments containing solitary LTRs were transcribed in normal germline cells (testicular parenchyma tissue). The differences in the expression of these clones in the germline tumor cells (seminoma) were observed.

Identification of paralogous HERV-K LTRs on human chromosomes 3, 4, 7 and 11 in regions containing clusters of olfactory receptor genes.

A locus harboring a human endogenous retroviral LTR (long terminal repeat) was mapped on the short arm of human chromosome 7 (7p22), and its evolutionary history was investigated. Sequences of two human genome fragments that were homologous to the LTR-flanking sequences were found in human genome databases: (1) an LTR-containing DNA fragment from region 3p13 of the human genome, which includes clusters of olfactory receptor genes and pseudogenes; and (2) a fragment of region 21q22.1 lacking LTR sequences. PCR analysis demonstrated that LTRs with highly homologous flanking sequences could be found in the genomes of human, chimp, gorilla, and orangutan, but were absent from the genomes of gibbon and New World monkeys. A PCR assay with a primer set corresponding to the sequence from human Chr 3 allowed us to detect LTR-containing paralogous sequences on human chromosomes 3, 4, 7, and 11. The divergence times for the LTR-flanking sequences on chromosomes 3 and 7, and the paralogous sequence on chromosome 21, were evaluated and used to reconstruct the order of duplication events and retroviral insertions. (1) An initial duplication event that occurred 14-17 Mya and before LTR insertion - produced two loci, one corresponding to that located on Chr 21, while the second was the ancestor of the loci on chromosomes 3 and 7. (2) Insertion of the LTR (most probably as a provirus) into this ancestral locus took place 13 Mya. (3) Duplication of the LTR-containing ancestral locus occurred 11 Mya, forming the paralogous modern loci on Chr 3 and 7.

[TAR-cloning of the short arm of human chromosome 7 in yeast and search for terminal sequences]. / TAR-klonirovanie korotkogo plecha khromosomy 7 cheloveka v drozhzhakh ipoisk terminal'nykh posledovatel'nostei.

A partial clone library of the short arm of human chromosome 7 was created in yeast artificial chromosomes (YAC) using TAR-cloning. The DNA of monochromosome somatic hybrid cells (mouse/human) RuRag 14-4-7-44 containing short arm human chromosome 7 was used for cloning. The clone library was screened for YACs with the human DNA; the mitotic stability of these YACs, the sizes of cloned fragments, and an independent clonal distribution in the chromosome were determined. Human YACs were tested for the presence of chromosome 7p telomeric sequences.