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REVERSIR platform for the tailored control of GalNAc-siRNA conjugate pharmacology

ABSTRACT: Primary mouse hepatocytes were treated with 1 nM TTR-siRNA and 10 nM scrambled or TTR-REVERSIR by in vitro free uptake for 24 h. RNA extracted with the Purelink RNA kit (ThermoFisher) was used for cDNA library preparation with the TruSeq Stranded Total RNA Library Prep Kit (Illumina) and sequenced on the NextSeq500 desktop sequencer (Illumina), all according to manufacturers’ instructions. Raw RNA-seq reads were filtered with minimal mean quality scores of 25 and minimal remaining length of 36, using fastq-mcf. Filtered reads were aligned to the Mus musculus genome (NCBIM37) using STAR with default parameters. Uniquely aligned reads were counted by featureCounts. Differential gene expression (DEG) analysis was performed by the R package DESeq2. Here, we report the rapid and potent reversal of GalNAc-siRNA-mediated RNAi activity in vivo with REVERSIR technology, which is based on short, synthetic high-affinity oligonucleotide complementary to the siRNA guide strand. The modular, sequence-specific nature of the REVERSIR platform has the potential to enhance the therapeutic profile of any long-acting GalNAc-siRNA conjugate to enable fine-tuned control of RNAi pharmacology

ORGANISM(S): Mus musculus

PROVIDER: GSE110620 | GEO | 2018/05/02

REPOSITORIES: GEO

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Project description:Purpose: To investigate the function of PLIN2 in human uterine leiomyoma tumorigenesis, we generated genome-wide transcription profile of leiomyoma cells after PLIN2 knockdown. Methods: Primary leiomyoma cells were isolated from fresh uterine leiomyoma tissues and transfected with PLIN2 small interfering RNA (siRNA; 100 nM, #4392422) or non-targeting siRNA (100 nM, #4390844; Ambion, Foster City, CA) for 72 hours. Total RNA was isolated using the RNeasy Mini kit and RNA-seq library was completed per the manufacturer’s instructions using the KAPA Stranded RNA-seq Kit with RiboErase (#KK8483; KAPA Biosystems, Wilmington, MA). cDNA libraries were sequenced as single-end, 75 base-length reads on an Illumina NextSeq 500 instrument (Illumina, San Diego, CA) with an average read count of 27 million reads per sample. The quality of DNA reads, in fastq format, was evaluated using FastQC. Adapters were trimmed, and reads of poor quality or aligning to rRNA sequences were filtered. The cleaned reads were aligned to the Homo sapiens genome (hg19) using STAR. Read counts for each gene were calculated using htseq-count in conjunction with a gene annotation file for hg19 obtained from University of California Santa Cruz (http://genome.ucsc.edu). Results: A total of 3877 genes were found to be differentially expressed, with 2557 genes found to be upregulated and 1320 genes found to be downregulated by PLIN2 knockdown. Gene Ontology analysis (DAVID, https://david.ncifcrf.gov) identified cellular component organization/biogenesis (P=1.4E-27) and metabolic processes (P=1.6E-10) as the top two biological processes of genes upregulated by PLIN2 depletion. Kyoto Encyclopedia of Genes and Genomes (KEGG; DAVID) pathway analysis identified enrichment of genes involved in the cell cycle, pyruvate metabolism, fatty acid metabolism and degradation, purine and pyrimidine metabolism, apoptosis, and extracellular matrix component organization after PLIN2 depletion. Conclusions: These findings provide support for a role of PLIN2 depletion in activating metabolic activity within leiomyoma cells.

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REVERSIR platform for the tailored control of GalNAc-siRNA conjugate pharmacology

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