Chemical Evolution of Amphiphilic Xenopeptides for Potentiated Cas9 Ribonucleoprotein Delivery

Yi Lin; Xianjin Luo; Tobias Burghardt; Sarah Dorrer; Miriam Höhn; Ernst Wagner; Ulrich Lächelt

doi:10.1021/jacs.3c01902

Chemical Evolution of Amphiphilic Xenopeptides for Potentiated Cas9 Ribonucleoprotein Delivery

Yi Lin, Xianjin Luo, Tobias Burghardt, Sarah Dorrer, Miriam Höhn, Ernst Wagner, Ulrich Lächelt

Department of Pharmaceutical Sciences

Publications: Contribution to journal › Article › Peer Reviewed

Abstract

The introduction of the CRISPR/Cas9 system in the form of Cas9/sgRNA ribonucleoproteins (RNP) is an efficient, straightforward strategy for genome editing, and potent RNP carriers are in high demand. Here, we report a series of artificial peptides based on novel ionizable amino acids that are able to deliver Cas9 RNP into cells very efficiently. Systematic variation of hydrophobic properties revealed a relationship between the xenopeptide logD_7.4 and genome editing potency. By correlating the physicochemical properties with biological activity, individual optima were found for different xenopeptide sequence architectures. The optimized amphiphilic carriers enable ∼88% eGFP knockout at an RNP dose of only 1 nM and up to 40% homology-directed repair (HDR) in eGFP/BFP switchable reporter cells by co-delivery with an ssDNA template. Mechanistic studies demonstrated that hydrophobically balanced xenopeptides are more resistant to ionic stress as well as concentration-dependent dissociation and promote endocytosis by both clathrin- and macropinocytosis-mediated pathways. The systematic study develops a versatile and adjustable carrier platform and highlights impactful structure-activity relationships, providing a new chemical guide for the design and optimization of nonviral Cas9 RNP nanocarriers.

Original language	English
Pages (from-to)	15171-15179
Number of pages	9
Journal	Journal of the American Chemical Society
Volume	145
Issue number	28
DOIs	https://doi.org/10.1021/jacs.3c01902
Publication status	Published - 19 Jul 2023

Funding

The authors acknowledge support by the UPGRADE (Unlocking Precision Gene Therapy) project that has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No 825825. This work was also supported by the German Research Foundation (DFG) SFB1032 (project-ID 201269156) sub-project B4. Y.L. and X.L. appreciate the fellowship of the China Scholarship Council that supports their Ph.D. studies. U.L. appreciates the support from the Galenus Foundation (Vienna, Austria). They thank Teoman Benli-Hoppe for performing MALDI-TOF mass spectrometry measurements. Figure 5A and Scheme S3 were created with BioRender.com .

Austrian Fields of Science 2012

301208 Pharmaceutical technology

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https://phaidra.univie.ac.at/o:2036325Licence: CC BY 4.0

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title = "Chemical Evolution of Amphiphilic Xenopeptides for Potentiated Cas9 Ribonucleoprotein Delivery",

abstract = "The introduction of the CRISPR/Cas9 system in the form of Cas9/sgRNA ribonucleoproteins (RNP) is an efficient, straightforward strategy for genome editing, and potent RNP carriers are in high demand. Here, we report a series of artificial peptides based on novel ionizable amino acids that are able to deliver Cas9 RNP into cells very efficiently. Systematic variation of hydrophobic properties revealed a relationship between the xenopeptide logD7.4 and genome editing potency. By correlating the physicochemical properties with biological activity, individual optima were found for different xenopeptide sequence architectures. The optimized amphiphilic carriers enable ∼88\% eGFP knockout at an RNP dose of only 1 nM and up to 40\% homology-directed repair (HDR) in eGFP/BFP switchable reporter cells by co-delivery with an ssDNA template. Mechanistic studies demonstrated that hydrophobically balanced xenopeptides are more resistant to ionic stress as well as concentration-dependent dissociation and promote endocytosis by both clathrin- and macropinocytosis-mediated pathways. The systematic study develops a versatile and adjustable carrier platform and highlights impactful structure-activity relationships, providing a new chemical guide for the design and optimization of nonviral Cas9 RNP nanocarriers.",

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AU - Wagner, Ernst

AU - Lächelt, Ulrich

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AB - The introduction of the CRISPR/Cas9 system in the form of Cas9/sgRNA ribonucleoproteins (RNP) is an efficient, straightforward strategy for genome editing, and potent RNP carriers are in high demand. Here, we report a series of artificial peptides based on novel ionizable amino acids that are able to deliver Cas9 RNP into cells very efficiently. Systematic variation of hydrophobic properties revealed a relationship between the xenopeptide logD7.4 and genome editing potency. By correlating the physicochemical properties with biological activity, individual optima were found for different xenopeptide sequence architectures. The optimized amphiphilic carriers enable ∼88% eGFP knockout at an RNP dose of only 1 nM and up to 40% homology-directed repair (HDR) in eGFP/BFP switchable reporter cells by co-delivery with an ssDNA template. Mechanistic studies demonstrated that hydrophobically balanced xenopeptides are more resistant to ionic stress as well as concentration-dependent dissociation and promote endocytosis by both clathrin- and macropinocytosis-mediated pathways. The systematic study develops a versatile and adjustable carrier platform and highlights impactful structure-activity relationships, providing a new chemical guide for the design and optimization of nonviral Cas9 RNP nanocarriers.

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Chemical Evolution of Amphiphilic Xenopeptides for Potentiated Cas9 Ribonucleoprotein Delivery

Abstract

Funding

Austrian Fields of Science 2012

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