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<p>Prime editing is a versatile genome editing technology that circumvents the need for DNA double-strand break formation and homology-directed repair, making it particularly suitable for in vivo correction of pathogenic mutations. Here we developed liver-specific prime editing approaches with temporally restricted prime editor (PE) expression. We first established a dual-delivery approach where the prime editor guide RNA is continuously expressed from adeno-associated viral vectors and only the PE is transiently delivered as nucleoside-modified mRNA encapsulated in lipid nanoparticles (LNP). This strategy achieved 26.2% editing with PEmax and 47.4% editing with PE7 at the <i>Dnmt1</i> locus using a single 2 mg kg<sup>−1</sup> dose of mRNA–LNP. When targeting the pathogenic <i>Pah</i><sup><i>enu2</i></sup> mutation in a phenylketonuria mouse model, gene correction rates reached 4.3% with PEmax and 20.7% with PE7 after three doses of 2 mg kg<sup>−1</sup> mRNA–LNP, effectively reducing blood <span>l</span>-phenylalanine levels from over 1,500 µmol l<sup>−1</sup> to below the therapeutic threshold of 360 µmol l<sup>−1</sup>. Encouraged by the high efficiency of PE7, we next explored a simplified approach where PE7 mRNA was co-delivered with synthetic prime editor guide RNAs encapsulated in LNP. This strategy yielded 35.9% editing after two doses of RNA–LNP at the <i>Dnmt1</i> locus and 8.0% editing after three doses of RNA–LNP at the <i>Pah</i><sup><i>enu2</i></sup> locus, again reducing <span>l</span>-phenylalanine levels below 360 µmol l<sup>−1</sup>. These findings highlight the therapeutic potential of mRNA–LNP-based prime editing for treating phenylketonuria and other genetic liver diseases, offering a scalable and efficient platform for future clinical translation.</p>

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Treatment of a metabolic liver disease in mice with a transient prime editing approach

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年5月20日

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  • Tanja Rothgangl,
  • András Tálas,
  • Eleonora I. Ioannidi,
  • Yanik Weber,
  • Desirée Böck,
  • Mai Matsushita,
  • Elina Andrea Villiger,
  • Lukas Schmidheini,
  • Woohyun J. Moon,
  • Paulo J. C. Lin,
  • Steven H. Y. Fan,
  • Kim F. Marquart,
  • Cornelia Schwerdel,
  • Nicole Rimann,
  • Erica Faccin,
  • Lukas Villiger,
  • Hiromi Muramatsu,
  • Máté Vadovics,
  • Alessio Cremonesi,
  • Péter István Kulcsár,
  • Beat Thöny,
  • Manfred Kopf,
  • Johannes Häberle,
  • Norbert Pardi,
  • Ying K. Tam,
  • Gerald Schwank

摘要

Prime editing is a versatile genome editing technology that circumvents the need for DNA double-strand break formation and homology-directed repair, making it particularly suitable for in vivo correction of pathogenic mutations. Here we developed liver-specific prime editing approaches with temporally restricted prime editor (PE) expression. We first established a dual-delivery approach where the prime editor guide RNA is continuously expressed from adeno-associated viral vectors and only the PE is transiently delivered as nucleoside-modified mRNA encapsulated in lipid nanoparticles (LNP). This strategy achieved 26.2% editing with PEmax and 47.4% editing with PE7 at the Dnmt1 locus using a single 2 mg kg−1 dose of mRNA–LNP. When targeting the pathogenic Pahenu2 mutation in a phenylketonuria mouse model, gene correction rates reached 4.3% with PEmax and 20.7% with PE7 after three doses of 2 mg kg−1 mRNA–LNP, effectively reducing blood l-phenylalanine levels from over 1,500 µmol l−1 to below the therapeutic threshold of 360 µmol l−1. Encouraged by the high efficiency of PE7, we next explored a simplified approach where PE7 mRNA was co-delivered with synthetic prime editor guide RNAs encapsulated in LNP. This strategy yielded 35.9% editing after two doses of RNA–LNP at the Dnmt1 locus and 8.0% editing after three doses of RNA–LNP at the Pahenu2 locus, again reducing l-phenylalanine levels below 360 µmol l−1. These findings highlight the therapeutic potential of mRNA–LNP-based prime editing for treating phenylketonuria and other genetic liver diseases, offering a scalable and efficient platform for future clinical translation.