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| Status |
Public on Dec 30, 2019 |
| Title |
Small RNA sequencing of mouse muscle and heart samples after small hairpin RNA delivery |
| Organism |
Mus musculus |
| Experiment type |
Non-coding RNA profiling by high throughput sequencing
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| Summary |
RNA interference (RNAi) is a promising gene therapy strategy for targeting dominant mutations. This therapy leverages small hairpin RNAs (shRNAs) to knock down gene expression; however, delivery of too much shRNA can disrupt the processing of endogenous microRNAs (miRNAs), and lead to toxicity. In this study, we sought to understand the effect that excessive shRNAs have on muscle miRNAs by transducing several constructs in mice and performing small RNA sequencing on their muscle and liver tissues. We found that shRNA expression was highest in the heart, and that when shRNAs accumulated to about 27% of total small RNAs, mice experienced substantial cardiomyopathy. At this level in the heart, shRNAs in other muscle tissues were only ~1.8-7.6% of total miRNAs. Regardless of treatment, the predominant heart microRNAs remained stable across samples, while the lower-expressed miR-451 – one of the only microRNAs processed in a Dicer-independent manner – changed in direct correlation with shRNA level and toxicity. Our data suggest that certain muscle miRNAs compete with exogenous shRNAs, leading to the observed cardiomyopathy, and that the mechanism of cardiotoxicity in these mice in response shRNA treatment was in contrast to what has been previously shown in the liver. Quantifying microRNA profiles after excessive shRNA delivery illuminates the host response to rAAV-shRNA, allowing for safer and more robust therapeutic gene knockdown, and gives us insight into the basic functions of muscle microRNAs.
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| Overall design |
Small RNA sequencing of mouse liver and five muscle tissues (Diaphragm, Heart, Gastrocnemius, Quadriceps and Tibialis Anterior) in biological triplicate in three groups: uninjected mice, those with small hairpin RNAs (shRNAs) against the Beta-galactosidase gene with a 19nt stem or 21nt stem. Tissues were harvested 2 weeks, 6 weeks or 12 weeks after adeno-associated virus 6 delivery (AAV6-shRNA). All conditions had a minimum of 3 samples.
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| Contributor(s) |
Valdmanis PN, Course MM |
| Citation(s) |
31927330 |
| Submission date |
Apr 16, 2019 |
| Last update date |
Jan 27, 2020 |
| Contact name |
Paul Valdmanis |
| E-mail(s) |
paulnv@uw.edu
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| Phone |
206-221-8059
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| Organization name |
University of Washington
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| Department |
Medicine
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| Lab |
Paul Valdmanis
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| Street address |
1705 NE Pacific St, HSB J-309
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| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98115-7720 |
| Country |
USA |
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| Platforms (2) |
| GPL16417 |
Illumina MiSeq (Mus musculus) |
| GPL21103 |
Illumina HiSeq 4000 (Mus musculus) |
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| Samples (86)
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| Relations |
| BioProject |
PRJNA533108 |
| SRA |
SRP192739 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE129896_muscle_miRNA_reads.txt.gz |
57.8 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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