Membrane permeabilizing amphiphilic peptide delivers recombinant transcription factor and CRISPR-Cas9/Cpf1 ribonucleoproteins in hard-to-modify cells.

Thomas Del’Guidice,Alain Garnier,Jean-Pascal Lepetit-Stoffaes,Louis-Jean Bordeleau,David Guay,Coraline Lauvaux,Jessica Trottier,Joannie Roberge,Denis-Claude Roy,Vibhuti Dave,Xavier Barbeau,Vanessa Théberge,Bruno Gaillet,Alfred S Lewin

doi:10.1371/journal.pone.0195558

Thomas Del’Guidice, Alain Garnier + Show 12 more

Open Access

https://doi.org/10.1371/journal.pone.0195558

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Abstract

Delivery of recombinant proteins to therapeutic cells is limited by a lack of efficient methods. This hinders the use of transcription factors or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) ribonucleoproteins to develop cell therapies. Here, we report a soluble peptide designed for the direct delivery of proteins to mammalian cells including human stem cells, hard-to-modify primary natural killer (NK) cells, and cancer cell models. This peptide is composed of a 6x histidine-rich domain fused to the endosomolytic peptide CM18 and the cell penetrating peptide PTD4. A less than two-minute co-incubation of 6His-CM18-PTD4 peptide with spCas9 and/or asCpf1 CRISPR ribonucleoproteins achieves robust gene editing. The same procedure, co-incubating with the transcription factor HoxB4, achieves transcriptional regulation. The broad applicability and flexibility of this DNA- and chemical-free method across different cell types, particularly hard-to-transfect cells, opens the way for a direct use of proteins for biomedical research and cell therapy manufacturing.

Highlights

Current viral and non-viral intracellular transfer methods mainly rely on the ex vivo delivery of foreign genetic material
Amongst PTD4/CM18-based peptide variants (S1 Table), the EV-Score revealed that linking a domain with 6 consecutive histidines to the CM18-PTD4, compared to 0, 3, 9 or 12
The co-delivery of GFP-NLS and HoxB4 with 6His-CM18-PTD4 resulted in GFP nuclear signal in 56.4% of cells and a HoxB4-mediated gene transcriptional modulation around 4 times higher than in the control condition (Fig 2J and 2K). These results suggest that 6His-CM18-PTD4 peptide can be used to regulate gene transcription by delivering recombinant transcription factors

Summary

Introduction

Current viral and non-viral intracellular transfer methods mainly rely on the ex vivo delivery of foreign genetic material. We report a peptide comprising both a CPP and an ELD that mediates the rapid, safe, and efficient cytosolic delivery of functional proteins to 20 mammalian cell types, including human stem cells, human primary cells and cancer cell models To achieve these delivery results, we fused the CPP Human Immunodeficiency Virus (HIV)-TAT variant PTD4 (YARAAARQARA) [19], with the cationic amphiphilic α-helical ELD CM18 (KWKLFKKIGAVL KVLTTG) [13]. Protein uptake could be performed in less than 2 minutes or maintained for hours and days This soluble, chemical-free and easy-to-use peptide opens new avenues to expand, differentiate, reprogram or edit therapeutic cells by the use of transcription factors, CRISPR RNP or any recombinant proteins

Materials and methods

Experimental methods

Results and discussion