Intein-mediated cytoplasmic reconstitution of a split toxin enables selective cell ablation in mixed populations and tumor xenografts

Abstract

The application of proteinaceous toxins for cell ablation is limited by their high on- and off-target toxicity, severe side effects, and a narrow therapeutic window. The selectivity of targeting can be improved by intein-based toxin reconstitution from two dysfunctional fragments provided their cytoplasmic delivery via independent, selective pathways. While the reconstitution of proteins from genetically encoded elements has been explored, exploiting cell-surface receptors for boosting selectivity has not been attained. We designed a robust splitting algorithm and achieved reliable cytoplasmic reconstitution of functional diphtheria toxin from engineered intein-flanked fragments upon receptor-mediated delivery of one of them to the cells expressing the counterpart. Retargeting the delivery machinery toward different receptors overexpressed in cancer cells enables selective ablation of specific subpopulations in mixed cell cultures. In a mouse model, the transmembrane delivery of a split-toxin construct potently inhibits the growth of xenograft tumors expressing the split counterpart. Receptor-mediated delivery of engineered split proteins provides a platform for precise therapeutic and experimental ablation of tumors or desired cell populations while also greatly expanding the applicability of the intein-based protein transsplicing.

Inteins as Drug Targets and Therapeutic Tools

“…In order to overcome this issue, a cysteine-less split intein was designed and active at ambient temperatures and in the absence of reducing agents, without requiring a denaturation step (Bhagawati et al, 2019;Basturea, 2021). The point of splitting of the target protein to fuse with split intein fragments is also important to get active protein after trans-splicing (Purde et al, 2020). Care must be taken to expose the least hydrophobic residues.…”

“…(A) Large gene delivery in gene therapy ( Tornabene et al, 2019 ; Lim et al, 2020 ). (B) Intein mediated toxin reconstitution inside the cells for selective cell killing in tumor therapy ( Purde et al, 2020 ). (C) Development of a BSL2 cell culture model for SARS-COV-2 in CaCo2 cells ( Ju et al, 2021 ).…”

“…Split intein was used to reconstitute a toxin inside selected cells enabling selective cell killing in mixed populations and tumor xenografts ( Purde et al, 2020 ). The Diphtheria toxin catalytic domain (DTA) was split into two, with each fused to split intein from N. punctiforme .…”

“…Split-toxinN encoding DNA is delivered by transfection/viral transduction, while recombinant split toxin C is delivered via a specific receptor by the anthrax toxin translocation system. The active toxin will be formed intracellularly by intein-mediated trans-splicing of two split-toxin parts ( Purde et al, 2020 ) ( Figure 2B ). The delivery of split toxin via cell surface-specific receptors will help in the killing of specific cell populations.…”

Reactive Chlorine Species Reversibly Inhibit DnaB Protein Splicing in Mycobacteria

“…The ability of inteins to rearrange or cleave peptide bonds in a controlled manner has been exploited in numerous biotechnological applications, including protein purification, segmental isotope labeling, formation of cyclic peptides, incorporation of nonnatural modules into proteins, fabrication of protein arrays, sensor development, imaging, and regulation of protein function in vivo ( 8 , 9 ). Additionally, new intein-based technologies frequently emerge ( 10 – 13 ). Given the useful chemistry performed by inteins, as well as the ability to leave no trace in the final product, the potential of these elements in protein engineering is tremendous.…”