Downregulation of human CCR5 gene expression with artificial microRNAs

Глазкова, Д. В.; Ветчинова, А. С.; Bogoslovskaya, E. V.; Zhogina, Y. A.; Markelov, Mikhail L.; Shipulin, German A.

doi:10.1134/s0026893313030035

Cited by 8 publications

(8 citation statements)

References 28 publications

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“…Targeting this site with sh1005 efficiently suppressed CCR5 expression and exhibited low toxicity [ 9 ]. Three new amiRNAs were created targeting these regions of CCR5, mic1001, mic1002, and mic1001lg ( Figure 1 a), which differed in their lengths of antisense strands by 20, 24, and 28 bp, respectively ( Figure 1 b), as this length can significantly influence amiRNA efficiency [ 8 , 17 ]. These amiRNAs were included in the LTR VECT lentiviral vector [ 22 ] with the puromycin resistance marker.…”

Section: Resultsmentioning

confidence: 99%

“…To test the silencing activity of the new amiRNAs, we used HT1080 CCR5-EGFP indicator cells with an imbedded transgene encoding the CCR5-EGFP chimeric protein [ 17 ]. Downregulation of CCR5-EGFP expression by amiRNAs against CCR5 can be easily quantified by detecting decreased EGFP fluorescence.…”

Section: Resultsmentioning

confidence: 99%

“…Primary CD4 + lymphocytes were activated by CD3/CD28 Dynabeads Human T-Activator (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and cultured in X-VIVO15 medium (Lonza, Basel, Switzerland) containing 100 IU/mL interleukin-2 (Biotech, St. Petersburg, Russia). HT1080 CCR5-EGFP cells [ 17 ] were cultured in Minimum Essential Medium supplemented with 10% FBS. Then, all were cultured at 37 °C and 5% CO 2 .…”

Section: Methodsmentioning

confidence: 99%

“…Pseudovirus particle preparation and infectious titer determination were performed according to a previously described protocol [ 17 ]. Briefly, HEK 293FT cells were cotransfected with the lentiviral vector plasmids and Lenti-X HT Packaging mix (Clontech, Mountain View, CA, USA).…”

Section: Methodsmentioning

confidence: 99%

“…Each construct was tested for its capacity to silence CCR5. One of them, the mic13lg + mic13lg construct, which contained two identical amiRNAs, showed the best results [ 17 ]. However, tandem repeat sequences in lentiviral constructs can potentially lead to deletions in the vector [ 18 , 19 ].…”

Section: Introductionmentioning

confidence: 99%

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Optimization of Polycistronic Anti-CCR5 Artificial microRNA Leads to Improved Accuracy of Its Lentiviral Vector Transfer and More Potent Inhibition of HIV-1 in CD4+ T-Cells

Urusov

Глазкова

Omelchenko

et al. 2018

Cells

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C-C chemokine receptor type 5 (CCR5) is utilized by human immunodeficiency virus (HIV) as a co-receptor for cell entry. Suppression of the CCR5 gene by artificial microRNAs (amiRNAs) could confer cell resistance. In previous work, we created a lentivector that encoded the polycistron of two identical amiRNAs that could effectively suppress CCR5. However, tandem repeats in lentiviral vectors led to deletions of the repeated sequences during reverse transcription of the vector RNA. To solve this problem, we have created a new amiRNA against CCR5, mic1002, which has a different microRNA scaffold and targets a different sequence. Replacing one of the two identical tandem amiRNAs in the polycistron with the mic1002 amiRNA increased the accuracy of its lentiviral vector transfer while retaining its ability to effectively suppress CCR5. A lentiviral vector containing two heterogenic amiRNAs significantly inhibited HIV replication in a vector-transduced human CD4+ lymphocyte culture.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Optimization of Polycistronic Anti-CCR5 Artificial microRNA Leads to Improved Accuracy of Its Lentiviral Vector Transfer and More Potent Inhibition of HIV-1 in CD4+ T-Cells

Urusov

Глазкова

Omelchenko

et al. 2018

Cells

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Problems and Prospects of Gene Therapy Against HIV

Schneider¹,

Wagner²,

Davydova³

et al. 2014

Pharm Chem J

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Optimization of Critical Hairpin Features Allows miRNA-based Gene Knockdown Upon Single-copy Transduction

Myburgh

Cherpin

Schlaepfer

et al. 2014

Molecular Therapy - Nucleic Acids

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Gene knockdown using micro RNA (miRNA)-based vector constructs is likely to become a prominent gene therapy approach. It was the aim of this study to improve the efficiency of gene knockdown through optimizing the structure of miRNA mimics. Knockdown of two target genes was analyzed: CCR5 and green fluorescent protein. We describe here a novel and optimized miRNA mimic design called mirGE comprising a lower stem length of 13 base pairs (bp), positioning of the targeting strand on the 5′ side of the miRNA, together with nucleotide mismatches in upper stem positions 1 and 12 placed on the passenger strand. Our mirGE proved superior to miR-30 in four aspects: yield of targeting strand incorporation into RNA-induced silencing complex (RISC); incorporation into RISC of correct targeting strand; precision of cleavage by Drosha; and ratio of targeting strand over passenger strand. A triple mirGE hairpin cassette targeting CCR5 was constructed. It allowed CCR5 knockdown with an efficiency of over 90% upon single-copy transduction. Importantly, single-copy expression of this construct rendered transduced target cells, including primary human macrophages, resistant to infection with a CCR5-tropic strain of HIV. Our results provide new insights for a better knockdown efficiency of constructs containing miRNA. Our results also provide the proof-of-principle that cells can be rendered HIV resistant through single-copy vector transduction, rendering this approach more compatible with clinical applications.

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Downregulation of human CCR5 gene expression with artificial microRNAs

Cited by 8 publications

References 28 publications

Optimization of Polycistronic Anti-CCR5 Artificial microRNA Leads to Improved Accuracy of Its Lentiviral Vector Transfer and More Potent Inhibition of HIV-1 in CD4+ T-Cells

Optimization of Polycistronic Anti-CCR5 Artificial microRNA Leads to Improved Accuracy of Its Lentiviral Vector Transfer and More Potent Inhibition of HIV-1 in CD4+ T-Cells

Problems and Prospects of Gene Therapy Against HIV

Optimization of Critical Hairpin Features Allows miRNA-based Gene Knockdown Upon Single-copy Transduction

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