RESUMEN
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.
Asunto(s)
Infecciones por VIH , VIH-1 , Ratones , Humanos , Animales , Infecciones por VIH/tratamiento farmacológico , VIH-1/genética , Células Madre Hematopoyéticas , Modelos Animales de Enfermedad , Federación de Rusia , Ratones SCIDRESUMEN
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.
RESUMEN
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.
Asunto(s)
Enzima Convertidora de Angiotensina 2 , COVID-19 , Modelos Animales de Enfermedad , Ratones , SARS-CoV-2 , Enzima Convertidora de Angiotensina 2/genética , Animales , Humanos , Ratones TransgénicosRESUMEN
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.
Asunto(s)
Vectores Genéticos , Ubiquitina-Proteína Ligasas , Proteínas Portadoras/genética , Vectores Genéticos/genética , Células HEK293 , Humanos , Lentivirus/genética , Transducción Genética , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
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.
Asunto(s)
Terapia Genética , Infecciones por VIH , Proteínas de Motivos Tripartitos/genética , Ubiquitina-Proteína Ligasas/genética , Factores de Restricción Antivirales , Infecciones por VIH/genética , Infecciones por VIH/terapia , HumanosRESUMEN
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.
Asunto(s)
Linfocitos T CD4-Positivos , Vectores Genéticos , Infecciones por VIH , VIH-1/fisiología , MicroARNs , Receptores CCR5 , Proteínas Recombinantes de Fusión , Replicación Viral , Factores de Restricción Antivirales , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/patología , Linfocitos T CD4-Positivos/virología , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Células HEK293 , Infecciones por VIH/genética , Infecciones por VIH/metabolismo , Infecciones por VIH/terapia , Humanos , MicroARNs/biosíntesis , MicroARNs/genética , Receptores CCR5/biosíntesis , Receptores CCR5/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína LigasasRESUMEN
Transpositions of the gypsy retrotransposon in the Drosophila melanogaster genome are controlled by the flamenco locus, which is represented as an accumulation of defective copies of transposable elements. In the present work, genetic control by the flamenco locus of the transcriptional and transpositional activities of the Tirant retrotransposon from the gypsy group was studied. Tissue-specific expression of Tirant was detected in the tissues of ovaries in a strain mutant for the flamenco locus. Tirant was found to be transpositionally active in isogenic D. melanogaster strains mutant for the flamenco locus. The sites of two new insertions have been localized by the method of subtractive hybridization. It has been concluded from the results obtained that the flamenco locus is involved in the genetic control of Tirant transpositions.
Asunto(s)
Sitios Genéticos , Genoma de los Insectos , Mutación , Retroelementos/genética , Transcripción Genética/genética , Animales , Drosophila melanogaster , Especificidad de Órganos/genéticaRESUMEN
We studied the influence of the preconditioning of wheat germ (Triticum aestivum L.) with 0.4 microM 24-epibrassidinole (EB) on the growth and hormone status of plants under the influence of 2% NaCl. The preconditioning with EB promoted the lowering of the extent of the damaging influence of pickling on the growth of germs. The important contribution to the realization of the protective action of EB in the preconditioning of plants is probably that of its ability to lower the level of stress-induced abscisic acid accumulation and the decrease in the content of indole-acetic acid. At the same time, the cytokinin concentration in plants preconditioned with EB under pickling was practically the same as in plants without stress. This fact combined with data about the ability of EB to induce the increase in cytokinin content in wheat, obtained before, allowed us to assume that the protective action of EB on plants is connected, first of all, with the prevention of the increase in level of hormones of cytokinin nature under pickling.