AAV amiRNAs with Engineered Scaffolds for Potent Gene Silencing In Vivo

The recent study by Militello et al., published in Scientific Reports, details the engineering and validation of novel artificial microRNA (amiRNA) scaffolds designed for highly efficient and precise gene silencing, particularly focusing on their robust and precise delivery via adeno-associated virus (AAV) vectors for sustained in vivo applications.

Background and Introduction

RNA interference (RNAi) is recognized as a powerful therapeutic strategy for targeting previously “undruggable” targets. However, existing siRNA-based therapies often face limitations such as transient effects and challenges in achieving precise, tissue-specific delivery. Artificial microRNAs (amiRNAs, also known as miRNA scaffolds) offer a more durable and precise approach to gene silencing. These amiRNAs are delivered as gene cassettes expressed from an RNA Polymerase II promoter, which allows for crucial tissue- and cell type-specific control of gene silencing and sustained effects compared to short interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs).

Adeno-associated virus (AAV) vectors have emerged as the leading platform for gene therapy delivery due to their excellent safety profile, low immunogenicity, and ability to mediate long-term transgene expression. The integration of engineered amiRNAs into AAV vectors enables sustained and targeted gene knockdown, which is especially valuable for neurological and genetic disorders. Among AAV serotypes, AAV9 is particularly valuable for central nervous system (CNS) applications due to its ability to cross the blood-brain barrier and transduce neurons efficiently. However, the effectiveness of RNAi-based therapies delivered via AAV depends critically on the design of the amiRNA scaffold, as suboptimal processing by the cellular RNAi machinery can lead to insufficient target knockdown or off-target effects.

Current amiRNA scaffolds (e.g., miR-155 or others) have several limitations:

• Inefficient processing by DROSHA/DICER complexes
• Variable guide strand loading into RISC
• Suboptimal silencing potency
• Potential for off-target effects

This study addressed these challenges through rational engineering of novel amiRNA scaffolds derived from highly expressed endogenous pri-miRNAs, incorporating specific sequence and structural modifications to enhance processing efficiency and precision. The research provides a comprehensive framework for developing next-generation RNAi therapeutics with improved efficacy and safety profiles.

Methods and Results

1. Design and Screening of Novel amiRNAs

The study’s design began with selecting highly expressed endogenous human pri-miRNAs to serve as scaffolds. These were then engineered to incorporate specific sequence determinants aimed at boosting the efficiency and precision of DROSHA and DICER processing. The modification strategies involved altering the stem base and loop regions, inserting motifs like GU dinucleotides and CHC bulges to encourage precise guide strand generation, and replacing the endogenous loop with a miR-30a loop for superior processing. To screen these novel amiRNAs, they were loaded with a guide targeting a fluorescent reporter in a U251-based cell line, and their silencing efficiency was assessed by measuring the reduction in reporter fluorescence following lentiviral delivery (Figure 1a and 1b).

Figure 1a and 1b. Novel amiRNA design and silencing efficiency reporter assay.

At the initial screening, various amiRNA constructs demonstrated significantly greater silencing efficiency, achieving 30-40% knockdown in cell lines compared to controls. Following this, the twelve top-performing amiRNAs underwent further validation across multiple biological replicates, utilizing a range of guide strands with varying potencies. The reporter assay, conducted in a U251-based cell line, consistently confirmed the superior silencing efficiency of these optimized amiRNAs.

Figure 1d and 1e. Silencing efficiency of selected amiRNAs; Reporter assay for novel amiRNAs loaded with guide sequences of varying potency.

2. Validation of Novel amiRNAs for Silencing Endogenous Genes

To validate knockdown efficiency, experiments targeted the endogenous CD9 transcript in 3T3 cells using amiRNAs delivered via lentiviral vector. Flow cytometry six days post-infection revealed a 14–52% increase in silencing efficiency compared to miRE and miR-155 (Fig. 2b). Further analysis of single-copy infected cells showed that eleven out of twelve amiRNAs had lower GFP transcript levels (Fig. 2c), confirming enhanced processing and increased silencing. The functionality of these amiRNAs was also confirmed when delivered by rAAV9 to U251-based mCherry reporter cells, demonstrating efficient silencing of the fluorescent target (Fig. 2d,e).

Figure 2b and 2c. Flow cytometry and qPCR analysis of GFP in 3T3 reporter cells.

Figure 2d and 2e. Schematic of rAAV9-mediated amiRNAs delivery in a reporter cell line and flow cytometry analysis of mCherry expression.

3. amiRNA In Vitro Processing in hIPSC-Derived Neurons

 This precision processing is crucial for decreasing off-targeting and ensuring biased loading onto RISC, thereby enhancing overall silencing efficiency. To demonstrate this, the researchers used human iPSC-derived NGN2 neurons, a therapeutically relevant model mimicking patient conditions to test processing accuracy. As depicted in Fig. 3a, rAAV9 encoding the novel amiRNAs with a guide against endogenous PTEN was used for transduction. qPCR data confirm strong PTEN mRNA silencing (Fig. 3b). Small-RNA sequencing reveals highly precise amiRNA processing, with over 98% of guide reads starting at expected positions (Fig. 3c), a finding confirmed across all novel amiRNAs (Fig. 3d). Guide strands are 100–1,000 times more abundant than passenger strands, indicating efficient RISC loading (Fig. 3e). Furthermore, silencing strength directly correlates with mature guide production (Fig. 3f). These results collectively underscore the importance of precise and efficient amiRNA processing for improved silencing strength and precision with minimal impact on cellular RNA.

Figure 3. rAAV9 delivery and precise amiRNA processing in human neurons.

4. In Vivo Validation Novel amiRNAs Delivered by rAAV9

To confirm in vitro results could be translated in vivo, a subset of novel amiRNAs with differing silencing efficacies (miRi-01, miRi-02, miRi-03) were delivered via rAAV9 into mice, with miRE as a comparison. These amiRNAs were engineered to target Ataxin-2.

rAAV9 vectors expressing amiRNAs were administered via intracerebroventricular (ICV) injection into the mice brain (Fig. 4a). Six weeks later, total RNA from cortexes showed minimal Ataxin-2 silencing with miRE and suboptimal miRi01. However, miRi02 and miRi03 yielded enhanced silencing, consistent with in vitro observations (Fig. 4b). Immunofluorescence further confirmed the novel amiRNAs’ superior in vivo silencing by quantifying ATAXIN2 expression in transduced cells (Fig. 4c,d). Crucially, small RNA-seq analysis demonstrated 1e2–1e3 fold greater guide strand production with precise processing in vivo (Fig. 4e,f), confirming the formation of a homogenous guide pool vital for therapeutic potential.

Figure 4. In Vivo Validation of amiRNA Function.

Conclusion

The study by Militello et al. successfully developed novel engineered amiRNAs with enhanced processing efficiency and precision, establishing a powerful platform for in vivo gene silencing. Using rAAV9 vectors for delivery, specifically via ICV injection, proved highly effective, achieving widespread and sustained gene silencing in the mouse brain. By overcoming the limitations of traditional RNAi methods—such as transient effects and inefficient delivery—this work lays foundational groundwork for future clinical applications of AAV-delivered amiRNAs in a wide array of diseases.

Reference:

https://www.nature.com/articles/s41598-025-07061-y