Effective and Accurate Gene Silencing by a Recombinant AAV-Compatible MicroRNA Scaffold

Xie, Jun; Tai, Phillip W. L.; Brown, Alexander; Gong, Sitang; Zhu, Sha; Wang, Yi; Li, Chengjian; Colpan, Cansu; Su, Qin; He, Ran; Ma, Hong; Li, Jia; Ye, Han-Qing; Ko, Jihye; Zamore, Phillip D.; Gao, Guangping

doi:10.1016/j.ymthe.2019.11.018

Cited by 26 publications

(39 citation statements)

References 38 publications

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“…The amiR against the mouse ctsk transcript was designed by using a custom Excel macro, which considers miR-33 scaffold design rules to generate optimized amiR cassettes. 28 DNA sequences for amiR-33-ctrl, amiR-33-rank-1 and -2, and amiR-33-ctsk-1 and -2 were synthesized as gBlocks and cloned into the intronic region of the pAAVsc-CB6-EGFP plasmid at PstI and BglII restriction enzyme sites. 43 Constructs were verified by Sanger sequencing.…”

Section: Methodsmentioning

confidence: 99%

“…Embedding the guide strand of a small silencing RNA into the miR-33-derived miRNA scaffold (amiR) limits short hairpin RNA (shRNA)-related toxicity, enables efficient gene knockdown, and reduces off-target silencing by 10fold compared to conventional shRNA constructs. 28 We therefore generated amiR cassettes targeting two different positions of rank (amiR-rank) or ctsk (amiR-ctsk) mRNA and then packaged them with AAV9 capsids (Figure 2A). Of note, the amiR cassette is embedded within the EGFP intronic region of the transgene cassette to track transduced cells or tissues.…”

Section: Raav9-mediated Gene Silencing In Ocs Increases Bone Mass In Micementioning

confidence: 99%

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Bone-Targeting AAV-Mediated Gene Silencing in Osteoclasts for Osteoporosis Therapy

Yang

Xie

Chaugule

et al. 2020

Molecular Therapy - Methods & Clinical Development

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Improper activity of bone-resorbing osteoclasts results in low bone density and deterioration of bone structure, which increase the risk of fractures. Anti-resorptive therapies targeting osteoclasts have proven effective in preserving bone mass, but these therapeutic agents lead to defective new bone formation and numerous potential side effects. In this study, we demonstrate that recombinant adeno-associated virus, serotype 9 (rAAV9) can deliver to osteoclasts an artificial microRNA (amiR) that silences expression of key osteoclast regulators, RANK (receptor activator for nuclear factor kB) and cathepsin K (rAAV9.amiR-rank, rAAV9.amiR-ctsk), to prevent bone loss in osteoporosis. As rAAV9 is highly effective for the transduction of osteoclasts, systemic administration of rAAV9 carrying amiR-rank or amiR-ctsk results in a significant increase of bone mass in mice. Furthermore, the bone-targeting peptide motif (Asp) 14 or (AspSerSer) 6 was grafted onto the AAV9-VP2 capsid protein, resulting in significant reduction of transgene expression in non-bone peripheral organs. Finally, systemic delivery of bone-targeting rAAV9.amiR-ctsk counteracts bone loss and improves bone mechanical properties in mouse models of postmenopausal and senile osteoporosis. Collectively, inhibition of osteoclast-mediated bone resorption via bone-targeting rAAV9-mediated silencing of ctsk is a promising gene therapy that can preserve bone formation and mitigate osteoporosis, while limiting adverse off-target effects.

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Section: Methodsmentioning

confidence: 99%

Section: Raav9-mediated Gene Silencing In Ocs Increases Bone Mass In Micementioning

confidence: 99%

Bone-Targeting AAV-Mediated Gene Silencing in Osteoclasts for Osteoporosis Therapy

Yang

Xie

Chaugule

et al. 2020

Molecular Therapy - Methods & Clinical Development

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“…Central bulges or kinks in the stem can prevent Ago2-mediated internal cleavage and trimming of shRNA precursors, as has been previously described in approaches to investigate Dicer activity or improve shRNA function (Vermeulen et al 2005;Vlassov et al 2007;Boudreau et al 2008;Wang et al 2008;Schopman et al 2010;Wu et al 2011;Jensen et al 2012;Matveeva et al 2012;Denise et al 2014;Fowler et al 2016;Watanabe et al 2016;Xie et al 2020). shRNA stem bulges have also been hypothesized to decrease thermodynamic pairing energy.…”

Section: Discussionmentioning

confidence: 91%

A functional screen for optimization of short hairpin RNA biogenesis and RISC loading

Sons¹,

Kaufmann²,

Hammond³

2020

Preprint

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Gene silencing via short hairpin mediated RNAi (shRNA) is a valuable experimental tool and has promise as a therapeutic strategy. Several shRNA platforms make use of the loop and flanking sequences from the endogenous microRNA (miRNAs) miR-30a or other miRNAs to provide an RNA structure for efficient and accurate biogenesis of the RNA trigger. However, the stem regions of these shRNAs are typically designed as perfect duplex structures which is an uncommon feature for endogenous miRNA precursors. A limitation of these designs is that shRNAs with perfect duplex stems undergo extensive stem cleavage analogous to the Dicer independent miRNA miR-451, destroying the shRNA trigger sequence that is present in the 3P arm. We employed an unbiased screen of > 9000 shRNA structures to identify features that prevent stem cleavage and promote canonical biogenesis and loading into the effector complex RISC. We find that a central stem bulge or kink reduces central stem cleavage and improves accuracy of Dicer processing. Furthermore, 9 -10 GC nucleotides in the guide strand improves shRNA efficiency. These design rules enable more effective shRNA tools and are compatible with existing sets of optimized guide/target sequences.

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“…In addition, most amiRNA stems are perfectly base‐paired, facilitating amiRNA design and analysis. However, the transcription of such highly structured constructs can be prematurely terminated, leading to the generation of inactive viral vectors (J. Xie et al, 2017, 2020).…”

Section: Structural Features Of Artificial Mirnasmentioning

confidence: 99%

“…In cultured cells and mice, the constructs effectively mediated gene silencing at a level similar to the corresponding shRNA. Furthermore, miR‐33‐based amiRNAs were accurately processed, generating many more guide strand than passenger strand (J. Xie et al, 2020). This backbone and the rules applied to its design were also used in trials of osteoporosis therapies.…”

Section: Artificial Mirna Scaffoldsmentioning

confidence: 99%

Artificial miRNAs as therapeutic tools: Challenges and opportunities

2021

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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

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Effective and Accurate Gene Silencing by a Recombinant AAV-Compatible MicroRNA Scaffold

Cited by 26 publications

References 38 publications

Bone-Targeting AAV-Mediated Gene Silencing in Osteoclasts for Osteoporosis Therapy

Bone-Targeting AAV-Mediated Gene Silencing in Osteoclasts for Osteoporosis Therapy

A functional screen for optimization of short hairpin RNA biogenesis and RISC loading

Artificial miRNAs as therapeutic tools: Challenges and opportunities

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