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Artificial miRNAs as therapeutic tools: Challenges and opportunities

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Abstract RNA interference (RNAi) technology has been used for almost two decades to study gene functions and in therapeutic approaches. It uses cellular machinery and small, designed RNAs in the form of synthetic small interfering RNAs (siRNAs) or vector‐based short hairpin RNAs (shRNAs), and artificial miRNAs (amiRNAs) to inhibit a gene of interest. Artificial miRNAs, known also as miRNA mimics, shRNA‐miRs, or pri‐miRNA‐like shRNAs have the most complex structures and undergo two‐step processing in cells to form mature siRNAs, which are RNAi effectors. AmiRNAs are composed of a target‐specific siRNA insert and scaffold based on a natural primary miRNA (pri‐miRNA). siRNAs serve as a guide to search for complementary sequences in transcripts, whereas pri‐miRNA scaffolds ensure proper processing and transport. The dynamics of siRNA maturation and siRNA levels in the cell resemble those of endogenous miRNAs; therefore amiRNAs are safer than other RNAi triggers. Delivered as viral vectors and expressed under tissue‐specific polymerase II (Pol II) promoters, amiRNAs provide long‐lasting silencing and expression in selected tissues. Therefore, amiRNAs are useful therapeutic tools for a broad spectrum of human diseases, including neurodegenerative diseases, cancers and viral infections. Recent reports on the role of sequence and structure in pri‐miRNA processing may contribute to the improvement of the amiRNA tools. In addition, the success of a recently initiated clinical trial for Huntington's disease could pave the way for other amiRNA‐based therapies, if proven effective and safe. This article is categorized under: RNA Processing > Processing of Small RNAs Regulatory RNAs/RNAi/Riboswitches > RNAi: Mechanisms of Action RNA in Disease and Development > RNA in Disease
Schematic representation of amiRNA expression cassettes. (a) AmiRNAs are mostly transcribed from Pol II promoters. AmiRNA can be expressed from their own promoters or can be located in the 3′ UTRs of protein‐coding genes (e.g., GFP) or embedded in an intron. (b) RNA Pol III promoters are also used for the expression of amiRNA constructs and RNA Pol III‐derived transcripts contain a 5′ppp and 3′ poly(U) tail resulting from the Pol III termination signal (usually 5–6 Ts). (c) Pol II promoters allow for the expression of multiple amiRNAs as a polycistronic transcript, individual transcripts or inducible expression systems
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Commonly used amiRNA scaffolds. Pri‐miR‐30a is the most frequently used amiRNA scaffold. It contains basal CNNC, UG and terminal GUG motifs (highlighted in green). The mature 22 nt miRNAs are derived from both the 5′ and 3′ ends, with the 5′ products predominating (from miRBase). Pri‐mir‐155 is the second most frequently used amiRNA scaffold. It does not contain sequence motifs that facilitate processing and generates miRNA from the 5′ end. DICER‐independent pri‐miR‐451 is processed by DROSHA, AGO2, and PARN, which does not generate a passenger strand
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Structural features of canonical pri‐miRNAs. Canonical pri‐miRNAs are composed of single‐stranded basal segments, a double‐stranded stem and terminal loop. During pri‐miRNA processing, the RNases DROSHA and DICER cleave the stem‐loop structure at specific positions to form the mature miRNA. The presence of sequence motifs (green), such as CNNC, GHG, UG, and UGU, help to position the cleavage site and enhance processing. The optimal length of the stem is ~35 ± 1 bp, and the stem is composed of the lower stem (13 bp), and upper stem (22 bp), which contains two bulge‐depleted regions
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Canonical and noncanonical miRNA biogenesis. MiRNA genes are transcribed mostly by RNA Pol II. Then, depending on the substrate three different miRNA biogenesis pathways lead to the production of mature miRNAs: (a) the DROSHA‐independent pathway, (b) canonical pathway, and (c) DICER‐independent pathway. (a) Mirtrons derived from short intronic hairpins are processed into pre‐miRNAs by spliceosomes and debranching enzymes, exported to the cytoplasm by XPO5 and cut by DICER into ~22 nt miRNAs. Endogenous short hairpin RNAs (endo‐shRNAs) are generated directly by transcription, and require XPO1 for nuclear transport. After DICER‐mediated cleavage, ~20 nt RNAs are produced, and only 3p‐ miRNAs are loaded onto AGO proteins. SnoRNA and tRNA‐derived miRNA are processed by DICER, transported by XPO5 and loaded onto AGO proteins. (b) Primary miRNA transcripts (pri‐miRNAs) undergo nuclear processing by the Microprocessor complex, which is composed of DROSHA, DGCR8, and SRSF3. RNase DROSHA cuts pri‐miRNA to the miRNA precursor (pre‐miRNA), which is transported to the cytoplasm by XPO5. In the cytoplasm, the pre‐miRNA is processed by DICER, its cofactor TRBP and protein kinase R‐activating protein (PACT). The RNAse III‐like enzyme DICER cleaves pre‐miRNAs and generates an imperfect ~22 nt miRNA duplex that is loaded onto an ARGONAUTE protein (AGO1‐4), forming the RNA‐induced silencing complex (RISC). AGO unwinds the duplex, the passenger strand is removed from the complex and the guide strand directs the activated RISC to the complementary target sequence in the mRNA. Depending on the level of complementarity, this results in translation inhibition, mRNA destabilization (partial) and/or target mRNA degradation by AGO2 (full complementarity). Nucleotides 2–8 from the 5′ end of the miRNA, called the “seed sequence,” are responsible for the specificity of the interactions. (c) MiR‐451 is the only known DICER‐independent miRNA. It is encoded in the same primary transcript as the DICER‐dependent miR‐144. DROSHA‐mediated cleavage of pri‐miRNA‐451 generates a hairpin with a short stem that is not recognized by DICER. The pre‐miRNA is loaded directly onto AGO2, which cleaves the 3′ strand of the stem and generates an ~30 nt product that is trimmed by poly(A)‐specific ribonuclease (PARN) to generate the mature miRNA. Translation initiation factor 1A (eIF1A) directly binds AGO2 and promotes miR‐451 biogenesis
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Comparison of synthetic (siRNA) and vector‐based (shRNA and amiRNA) RNAi triggers
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RNA in Disease and Development > RNA in Disease
Regulatory RNAs/RNAi/Riboswitches > RNAi: Mechanisms of Action
RNA Processing > Processing of Small RNAs

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