Efficacy, accumulation, and transcriptional profile of anti-HIV shRNAs expressed from human U6, 7SK, and H1 promoters
The expression of short hairpin RNAs (shRNAs) in cells has many potential therapeutic applications, including as a functional cure for HIV. The RNA polymerase III promoters H1, 7SK, and U6 have all been used to express shRNAs. However, there have been no direct and simultaneous comparisons of shRNA...
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doaj-5c5eb089f6c0434f859658bd9d510f3c2021-03-07T04:27:59ZengElsevierMolecular Therapy: Nucleic Acids2162-25312021-03-012310201034Efficacy, accumulation, and transcriptional profile of anti-HIV shRNAs expressed from human U6, 7SK, and H1 promotersRyan P. Goguen0Olivier Del Corpo1Camille M.G. Malard2Aïcha Daher3Sergio P. Alpuche-Lazcano4Michelle J. Chen5Robert J. Scarborough6Anne Gatignol7Virus-Cell Interactions Laboratory, Lady Davis Institute for Medical Research, Montréal, QC H3T 1E2, Canada; Department of Microbiology and Immunology, McGill University, Montréal, QC H3A 0G4, CanadaVirus-Cell Interactions Laboratory, Lady Davis Institute for Medical Research, Montréal, QC H3T 1E2, Canada; Department of Microbiology and Immunology, McGill University, Montréal, QC H3A 0G4, Canada; Division of Experimental Medicine, Division of Infectious Diseases, Department of Medicine, McGill University, Montréal, QC H3A 0G4, CanadaVirus-Cell Interactions Laboratory, Lady Davis Institute for Medical Research, Montréal, QC H3T 1E2, Canada; Department of Microbiology and Immunology, McGill University, Montréal, QC H3A 0G4, CanadaVirus-Cell Interactions Laboratory, Lady Davis Institute for Medical Research, Montréal, QC H3T 1E2, CanadaVirus-Cell Interactions Laboratory, Lady Davis Institute for Medical Research, Montréal, QC H3T 1E2, Canada; Division of Experimental Medicine, Division of Infectious Diseases, Department of Medicine, McGill University, Montréal, QC H3A 0G4, CanadaVirus-Cell Interactions Laboratory, Lady Davis Institute for Medical Research, Montréal, QC H3T 1E2, Canada; Department of Microbiology and Immunology, McGill University, Montréal, QC H3A 0G4, Canada; Division of Experimental Medicine, Division of Infectious Diseases, Department of Medicine, McGill University, Montréal, QC H3A 0G4, CanadaVirus-Cell Interactions Laboratory, Lady Davis Institute for Medical Research, Montréal, QC H3T 1E2, Canada; Department of Microbiology and Immunology, McGill University, Montréal, QC H3A 0G4, Canada; Corresponding author: Robert Scarborough, Virus-Cell Interactions Laboratory, Lady Davis Institute for Medical Research, Montréal, QC H3T 1E2, Canada.Virus-Cell Interactions Laboratory, Lady Davis Institute for Medical Research, Montréal, QC H3T 1E2, Canada; Department of Microbiology and Immunology, McGill University, Montréal, QC H3A 0G4, Canada; Division of Experimental Medicine, Division of Infectious Diseases, Department of Medicine, McGill University, Montréal, QC H3A 0G4, Canada; Corresponding author: Anne Gatignol, Virus-Cell Interactions Laboratory, Lady Davis Institute for Medical Research, Montréal, QC H3T 1E2, Canada.The expression of short hairpin RNAs (shRNAs) in cells has many potential therapeutic applications, including as a functional cure for HIV. The RNA polymerase III promoters H1, 7SK, and U6 have all been used to express shRNAs. However, there have been no direct and simultaneous comparisons of shRNA potency, expression level, and transcriptional profile between the promoters. We show that the 7SK and U6 promoters result in higher shRNA levels and potency compared to the H1 promoter but that in transduced T lymphocytes, higher expression levels can also lead to growth defects. We present evidence that Dicer cleavage of shRNAs is measured from the first base pair in the shRNA stem, rather than from the 5′ end as previously shown for structurally related microRNAs. As a result, guide-strand identity was unaffected by variations in 5′ transcription start sites among the different promoters, making expression levels the main determinant of shRNA potency. While all promoters generated shRNAs with variable start sites, the U6 promoter was the most accurate in using its intended +1 position. Our results have implications for the development of therapeutic small RNAs for gene therapy and for our understanding of how shRNAs are processed in cells.http://www.sciencedirect.com/science/article/pii/S2162253120304030HIV/AIDSRNA therapyRNA Polymerase III promotersshRNAshort haiprin RNAGene therapy |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Ryan P. Goguen Olivier Del Corpo Camille M.G. Malard Aïcha Daher Sergio P. Alpuche-Lazcano Michelle J. Chen Robert J. Scarborough Anne Gatignol |
spellingShingle |
Ryan P. Goguen Olivier Del Corpo Camille M.G. Malard Aïcha Daher Sergio P. Alpuche-Lazcano Michelle J. Chen Robert J. Scarborough Anne Gatignol Efficacy, accumulation, and transcriptional profile of anti-HIV shRNAs expressed from human U6, 7SK, and H1 promoters Molecular Therapy: Nucleic Acids HIV/AIDS RNA therapy RNA Polymerase III promoters shRNA short haiprin RNA Gene therapy |
author_facet |
Ryan P. Goguen Olivier Del Corpo Camille M.G. Malard Aïcha Daher Sergio P. Alpuche-Lazcano Michelle J. Chen Robert J. Scarborough Anne Gatignol |
author_sort |
Ryan P. Goguen |
title |
Efficacy, accumulation, and transcriptional profile of anti-HIV shRNAs expressed from human U6, 7SK, and H1 promoters |
title_short |
Efficacy, accumulation, and transcriptional profile of anti-HIV shRNAs expressed from human U6, 7SK, and H1 promoters |
title_full |
Efficacy, accumulation, and transcriptional profile of anti-HIV shRNAs expressed from human U6, 7SK, and H1 promoters |
title_fullStr |
Efficacy, accumulation, and transcriptional profile of anti-HIV shRNAs expressed from human U6, 7SK, and H1 promoters |
title_full_unstemmed |
Efficacy, accumulation, and transcriptional profile of anti-HIV shRNAs expressed from human U6, 7SK, and H1 promoters |
title_sort |
efficacy, accumulation, and transcriptional profile of anti-hiv shrnas expressed from human u6, 7sk, and h1 promoters |
publisher |
Elsevier |
series |
Molecular Therapy: Nucleic Acids |
issn |
2162-2531 |
publishDate |
2021-03-01 |
description |
The expression of short hairpin RNAs (shRNAs) in cells has many potential therapeutic applications, including as a functional cure for HIV. The RNA polymerase III promoters H1, 7SK, and U6 have all been used to express shRNAs. However, there have been no direct and simultaneous comparisons of shRNA potency, expression level, and transcriptional profile between the promoters. We show that the 7SK and U6 promoters result in higher shRNA levels and potency compared to the H1 promoter but that in transduced T lymphocytes, higher expression levels can also lead to growth defects. We present evidence that Dicer cleavage of shRNAs is measured from the first base pair in the shRNA stem, rather than from the 5′ end as previously shown for structurally related microRNAs. As a result, guide-strand identity was unaffected by variations in 5′ transcription start sites among the different promoters, making expression levels the main determinant of shRNA potency. While all promoters generated shRNAs with variable start sites, the U6 promoter was the most accurate in using its intended +1 position. Our results have implications for the development of therapeutic small RNAs for gene therapy and for our understanding of how shRNAs are processed in cells. |
topic |
HIV/AIDS RNA therapy RNA Polymerase III promoters shRNA short haiprin RNA Gene therapy |
url |
http://www.sciencedirect.com/science/article/pii/S2162253120304030 |
work_keys_str_mv |
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