[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.

[Protease and reverse transcriptase genetic polymorphism in HIV type 1 subtype A variants predominating in cis countries].

To define frequencies of drug resistance mutations among HIV-1 variants circulating within the territory of Russia, subtype A HIV-1 nucleotide sequences encoding protease and reverse transcriptase were analyzed. The analysis was carried out in 141 antiretroviral-naive individuals. Low frequency (less than 1%) of primary drug resistance mutations was shown. However, high frequencies of secondary mutations V77I in protease and A62V in RT (67% H 63%, respectively) linked to each other in most cases were observed. The HIV-1 isolates bearing both substitutions (MutV77I/A62V) were also characterized by the presence of several synonymous mutations, suggesting common origin for these viruses. HIV Biochip Hybridization microarray and/or Restriction fragment-length polymorphism analyses were performed to characterize gene pol polymorphism in additional 178 subtype A HIV-1 isolates. Among total 319 samples studied, Mutv77IA62V variant accounted for 56%, and was found to predominate in Russia in terms of both its geographical distribution and number of cases caused. Moreover, these viruses were prevalent in the regions known to have highest incidence of HIV-1 infection (Irkutsk, Samara, and Moscow regions). In addition, three other variants were found: viruses not containing the substitutions V77I or A62V, and variants bearing only one of them. Evolutional relationships between all four HIV-1 variants, as well as potential impact of the gene pol polymorphism on HIV-1 replicative fitness and drug resistance development are discussed.

[Antiretroviral therapy of acute HIV-infection]. / Antiretrovirusnaia terapiia ostroi VICh-infektsii.

AIM: To study efficacy of a short course of highly active antiretroviral therapy (HAART). MATERIAL AND METHODS: Acute HIV infection was verified in 16 infected subjected basing either on seroconversion (by immunoblotting) or a documented negative result 6-12 months before registration of a positive result for HIV antibodies. RESULTS: HAART was given to 12 patients (combivir + nevirapin). The number of CD+ lymphocytes (by median) 1 month after the treatment increased by 185 cells/mcl, 3 months after the treatment--by 215 cells/mcl. After 1-month therapy viral load (median) diminished by 2.02 log10 copy/ml, after 3 months--by 2.31 log10 copy/ml. 71% patients had HIV RNA under 400 copy/ml. Untreated patients showed changes neither in CD4+ lymphocytes number nor in viral load. The study continues. CONCLUSION: HAART is used at the stage of acute infection in the presence of psychic trauma provoked by establishment of HIV-infection diagnosis. Therefore, it is necessary to consult the patients for preparing them for treatment and to maintain compliance.

[Determination of HIV RNA concentration for evaluation of the efficacy of combined therapy of HIV infection]. / Opredelenie kontsentratsii RNK VICh dlia otsenkii éffektivnosti kombinirovannoi terapii VICh-infektsii.

Plasma HIV RNA (viral load) and count of CD4+ T cells were evaluated in 23 patients with HIV infection treated with invirase (1800 mg/day), zidovudine (600 mg/day), and zalcitabine (2.25 mg/day) for 6 months in order to evaluate the efficiency of antiretroviral therapy. Viral load was measured by AMPLICOR HIV-1 Monitor test. The reproducibility of HIV RNA measurements was in line with reported data (CV 15-41%), allowing highly accurate (15%) evaluation of RNA in a standard control sample provided by National Institute for Biological Standards and Control, Great Britain. Plasma HIV RNA concentration decreased to an undetectable level (below 400 RNA copies/ml plasma) after 6 months of treatment in 52.2% patients. In 17.4% the therapy failed, and in 30.4% it resulted in a reduction of viral load to > 1 lg, although HIV RNA was still detected in the plasma after 6-month therapy. The count of CD4+ T cells increased by 9.5%. Changes in the viral load outstripped changes in CD4+ cells. Viral load was in high correlation with the count of CD4+ lymphocytes: -0.53, p = 0.01 before treatment and -0.61, p = 0.002 after 6-month treatment.