Systematic Screening, Rational Development, and Initial Optimization of Efficacious RNA Silencing Agents for Human Rod Opsin Therapeutics.

PURPOSE: To systematically evaluate human rod opsin (hRHO) mRNA for potential target sites sensitive to posttranscriptional gene silencing (PTGS) by hammerhead ribozyme (hhRz) or RNA interference (RNAi) in human cells. To develop a comprehensive strategy to identify and optimize lead candidate agents for PTGS gene therapeutics. METHODS: In multidisciplinary RNA drug discovery, computational mRNA accessibility and in vitro experimental methods using reverse transcription-polymerase chain reaction (RT-PCR) were used to map accessibility in full-length hRHO transcripts. HhRzs targeted predicted accessible and inaccessible sites and were screened for cellular knockdown using a bicistronic reporter construct. Lead hhRz and RNAi PTGS agents were rationally optimized for target knockdown in human cells. RESULTS: Systematic screening of hRHO mRNA targeting agents resulted in lead candidate identification of a novel hhRz embedded in an RNA scaffold. Rational optimization strategies identified a minimal 725 hhRz as the most active agent. Recently identified tertiary accessory elements did not enhance activity. A 725-short-hairpin RNA (shRNA) agent exerts log-order knockdown. Silent modulation of the 725-hhRz target site in hRHO mRNA resulted in resistance to knockdown. CONCLUSIONS: Combining rational RNA drug design with cell-based screening allowed rapid identification of lead agents targeting hRHO. Optimization strategies identified the agent with highest intracellular activity. These agents have therapeutic potential in a mutation-independent strategy for adRP, or other degenerations where hRHO is a target. This approach can be broadly applied to any validated target mRNA, regardless of the disease. TRANSLATIONAL RELEVANCE: This work establishes a platform approach to develop RNA biologicals for the treatment of human disease.

Mitochondrial complex II regulates a distinct oxygen sensing mechanism in monocytes.

Mutations in mitochondrial complex II (succinate dehydrogenase; SDH) genes predispose to paraganglioma tumors that show constitutive activation of hypoxia responses. We recently showed that SDHB mRNAs in hypoxic monocytes gain a stop codon mutation by APOBEC3A-mediated C-to-U RNA editing. Here, we test the hypothesis that inhibition of complex II facilitates hypoxic gene expression in monocytes using an integrative experimental approach. By RNA sequencing, we show that specific inhibition of complex II by atpenin A5 in normoxic conditions mimics hypoxia and induces hypoxic transcripts as well as APOBEC3A-mediated RNA editing in human monocytes. Myxothiazol, a complex III inhibitor, has similar effects in normoxic monocytes. Atpenin A5 partially inhibits oxygen consumption, and neither hypoxia nor atpenin A5 in normoxia robustly stabilizes hypoxia-inducible factor (HIF)-1α in primary monocytes. Several earlier studies in transformed cell lines suggested that normoxic stabilization of HIF-1α explains the persistent expression of hypoxic genes upon complex II inactivation. On the contrary, we find that atpenin A5 antagonizes the stabilization of HIF-1α and reduces hypoxic gene expression in transformed cell lines. Accordingly, compound germline heterozygosity of mouse Sdhb/Sdhc/Sdhd null alleles blunts chronic hypoxia-induced increases in hemoglobin levels, an adaptive response mainly regulated by HIF-2α. In contrast, atpenin A5 or myxothiazol does not reduce hypoxia-induced gene expression or RNA editing in monocytes. These results reveal a novel role for mitochondrial respiratory inhibition in induction of the hypoxic transcriptome in monocytes and suggest that inhibition of complex II activates a distinct hypoxia signaling pathway in a cell-type specific manner.

The double-domain cytidine deaminase APOBEC3G is a cellular site-specific RNA editing enzyme.

APOBEC3G is a cytidine deaminase with two homologous domains and restricts retroelements and HIV-1. APOBEC3G deaminates single-stranded DNAs via its C-terminal domain, whereas the N-terminal domain is considered non-catalytic. Although APOBEC3G is known to bind RNAs, APOBEC3G-mediated RNA editing has not been observed. We recently discovered RNA editing by the single-domain enzyme APOBEC3A in innate immune cells. To determine if APOBEC3G is capable of RNA editing, we transiently expressed APOBEC3G in the HEK293T cell line and performed transcriptome-wide RNA sequencing. We show that APOBEC3G causes site-specific C-to-U editing of mRNAs from over 600 genes. The edited cytidines are often flanked by inverted repeats, but are largely distinct from those deaminated by APOBEC3A. We verified protein-recoding RNA editing of selected genes including several that are known to be involved in HIV-1 infectivity. APOBEC3G co-purifies with highly edited mRNA substrates. We find that conserved catalytic residues in both cytidine deaminase domains are required for RNA editing. Our findings demonstrate the novel RNA editing function of APOBEC3G and suggest a role for the N-terminal domain in RNA editing.

Hypoxia-inducible C-to-U coding RNA editing downregulates SDHB in monocytes.

Background. RNA editing is a post-transcriptional regulatory mechanism that can alter the coding sequences of certain genes in response to physiological demands. We previously identified C-to-U RNA editing (C136U, R46X) which inactivates a small fraction of succinate dehydrogenase (SDH; mitochondrial complex II) subunit B gene (SDHB) mRNAs in normal steady-state peripheral blood mononuclear cells (PBMCs). SDH is a heterotetrameric tumor suppressor complex which when mutated causes paraganglioma tumors that are characterized by constitutive activation of hypoxia inducible pathways. Here, we studied regulation, extent and cell type origin of SDHB RNA editing. Methods. We used short-term cultured PBMCs obtained from random healthy platelet donors, performed monocyte enrichment by cold aggregation, employed a novel allele-specific quantitative PCR method, flow cytometry, immunologic cell separation, gene expression microarray, database analysis and high-throughput RNA sequencing. Results. While the editing rate is low in uncultured monocyte-enriched PBMCs (average rate 2.0%, range 0.4%-6.3%, n = 42), it is markedly upregulated upon exposure to 1% oxygen tension (average rate 18.2%, range 2.8%-49.4%, n = 14) and during normoxic macrophage differentiation in the presence of serum (average rate 10.1%, range 2.7%-18.8%, n = 17). The normoxic induction of SDHB RNA editing was associated with the development of dense adherent aggregates of monocytes in culture. CD14-positive monocyte isolation increased the percentages of C136U transcripts by 1.25-fold in normoxic cultures (n = 5) and 1.68-fold in hypoxic cultures (n = 4). CD14-negative lymphocytes showed no evidence of SDHB editing. The SDHB genomic DNA remained wild-type during increased RNA editing. Microarray analysis showed expression changes in wound healing and immune response pathway genes as the editing rate increased in normoxic cultures. High-throughput sequencing of SDHB and SDHD transcripts confirmed the induction of C136U RNA editing in normoxic cultures but showed no additional verifiable coding edits. Analysis of SDHB RNA sequence data from 16 normal human tissues from the Illumina Body Map and from 45 samples representing 23 different cell types from the ENCODE projects confirmed the occurrence of site-specific C136U editing in whole blood (1.7%) and two primary CD14+ monocyte samples (1.9% and 2.6%). In contrast, the other cell types showed an average of 0.2% and 0.1% C136U editing rates in the two databases, respectively. Conclusions. These findings demonstrate that C-to-U coding RNA editing of certain genes is dynamically induced by physiologically relevant environmental factors and suggest that epigenetic downregulation of SDHB by site-specific RNA editing plays a role in hypoxia adaptation in monocytes.