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Improved induction of ribozyme-controlled AAV transgene via peptide-conjugated morpholino oligos

Gene Therapy volume 32, pages 80–82 (2025)Cite this article

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The Original Article was published on 12 February 2025

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Adeno-associated virus (AAV)-mediated transgene delivery has emerged as a cornerstone in the field of gene therapy [1, 2]. Due to its superior safety profile, clinical efficacy, and broad tissue tropism, AAV-based therapies have been successfully used for treating human genetic disorders, such as inherited blindness and spinal muscular atrophy [3, 4]. However, challenges such as the limited packaging capacity of AAV and the lack of temporal or dose control of transgene expression hinder AAV’s full clinical potential, particularly when contraindications and adverse effects may arise over time [3, 5]. Developing more versatile transgene delivery systems that enable post-delivery manipulation could broaden the scope of AAV-based therapies for a wider range of conditions. Tang et al. in this issue addressed this challenge by improving the T3H38 ribozyme-based RNA switch system in mice, which enabled efficient and durable induction of AAV transgene expression by systemically administering cell-penetrating peptide (CPP)-conjugated morpholino oligos [6].

Although the T3H38/v-M8 switch system enables efficient gene turnover with a dynamic range of up to 200-fold in mice, it has limitations, including effective function only after intramuscular, rather than intravenous, administration of the v-M8 morpholino oligo. Additionally, its short induction half-life may require frequent inducer dosing for ‘bio-factory’ gene therapy [9]. To overcome these challenges, Tang et al. [6] in this issue of Gene Therapy have replaced the octa-guanidine dendrimer in the v-M8 morpholino with a cell-penetrating peptide (CPP), a delivery moiety validated in mice, monkeys, and humans for systemic morpholino oligo administration [11, 12]. They tested two CPPs, either the B peptide (B-M8) or a fusion of the B peptide with a muscle-specific peptide (B-MSP-M8), to improve the delivery and efficacy of morpholino oligo, by assessing their ability to induce gene expression in a T3H38-regulated AAV transgene system. Intramuscular injection of all three morpholino oligos (v-M8, B-M8, and B-MSP-M8) efficiently induced expression of the firefly luciferase (Fluc) reporter. Notably, the B-MSP-M8 oligo led to the most sustained expression, with Fluc activity persisting for approximately five months and an induction half-life of 61 days, which is 7.6 times that of v-M8 and 1.7 times that of B-M8. The performance of B-MSP-M8 was consistent across both male and female mice, demonstrating its robust and gender-independent efficacy. The systemic administration of the oligos was also evaluated for AAV-Fluc induction following intravenous administration. With the v-M8 oligo, Fluc expression increased 9-fold. Although B-M8 turned out to be ineffective, the B-MSP-M8 oligo induced an 87-fold increase in Fluc expression, accompanied by an induction half-life of 33.5 days (Fig. 1). In conclusion, this study highlights that the B-MSP conjugate effectively induces AAV transgene expression in skeletal muscle through systemic delivery of the morpholino oligo inducer [6].

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Fig. 1: CCP-conjugated morpholino enables sustained induction of ribozyme-controlled AAV transgene.

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Funding

This work was supported by the National Natural Science Foundation of China grant (32171444 to BC).

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Authors and Affiliations

  1. The Fourth Affiliated Hospital, Zhejiang University School of Medicine, Yiwu, China

    Tianyi Cheng & Wei Zou

  2. Insititute of Translational Medicine, Zhejiang University, Hangzhou, China

    Tianyi Cheng & Wei Zou

  3. Department of Cell Biology, Zhejiang University School of Medicine, Hangzhou, China

    Tianyi Cheng & Baohui Chen

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  1. Tianyi Cheng
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  2. Baohui Chen
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  3. Wei Zou
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TC drafted the initial manuscript. BC and WZ edited the text and approved the final version.

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Correspondence to Baohui Chen or Wei Zou.

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Cheng, T., Chen, B. & Zou, W. Improved induction of ribozyme-controlled AAV transgene via peptide-conjugated morpholino oligos. Gene Ther 32, 80–82 (2025). https://doi.org/10.1038/s41434-025-00520-1

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