Amphiphilic shuttle peptide delivers base editor ribonucleoprotein to correct the CFTR R553X mutation in well-differentiated airway epithelial cells.

Base editing could correct nonsense mutations that cause cystic fibrosis (CF), but clinical development is limited by the lack of delivery methods that efficiently breach the barriers presented by airway epithelia. Here, we present a novel amphiphilic shuttle peptide based on the previously reported S10 peptide that substantially improved base editor ribonucleoprotein (RNP) delivery. Studies of the S10 secondary structure revealed that the alpha-helix formed by the endosomal leakage domain (ELD), but not the cell penetrating peptide (CPP), was functionally important for delivery. By isolating and extending the ELD, we created a novel shuttle peptide, termed S237. While S237 achieved lower delivery of green fluorescent protein, it outperformed S10 at Cas9 RNP delivery to cultured human airway epithelial cells and to pig airway epithelia in vivo, possibly due to its lower net charge. In well-differentiated primary human airway epithelial cell cultures, S237 achieved a 4.6-fold increase in base editor RNP delivery, correcting up to 9.4% of the cystic fibrosis transmembrane conductance regulator (CFTR) R553X allele and restoring CFTR channel function close to non-CF levels. These findings deepen the understanding of peptide-mediated delivery and offer a translational approach for base editor RNP delivery for CF airway disease.

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

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.

Activation of Phenyl 4-(2-Oxo-3-alkylimidazolidin-1-yl)benzenesulfonates Prodrugs by CYP1A1 as New Antimitotics Targeting Breast Cancer Cells.

Prodrug-mediated utilization of the cytochrome P450 (CYP) 1A1 to obtain the selective release of potent anticancer products within cancer tissues is a promising approach in chemotherapy. We herein report the rationale, preparation, biological evaluation, and mechanism of action of phenyl 4-(2-oxo-3-alkylimidazolidin-1-yl)benzenesulfonates (PAIB-SOs) that are antimicrotubule prodrugs activated by CYP1A1. Although PAIB-SOs are inert in most cells tested, they are highly cytocidal toward several human breast cancer cells, including hormone-independent and chemoresistant types. PAIB-SOs are N-dealkylated into cytotoxic phenyl 4-(2-oxo-3-imidazolidin-1-yl)benzenesulfonates (PIB-SOs) in CYP1A1-positive cancer cells, both in vitro and in vivo. In conclusion, PAIB-SOs are novel chemotherapeutic prodrugs with no equivalent among current antineoplastics and whose selective action toward breast cancer is tailored to the characteristic pattern of CYP1A1 expression observed in a large percentage of human breast tumors.