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bioRxiv · 10.1101/2024.09.27.615476

Earlier endosomal escape improves the catalytic activity of delivered enzyme cargo

Vazquez Maldonado, A. L.·Chen, T.·Rodriguez, D.·Zoltek, M.·Schepartz, A.
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Abstract

There is enormous interest in strategies to efficiently traffic biologics-proteins, nucleic acids, and complexes thereof-into the mammalian cell cytosol and internal organelles. Not only must these materials reach the appropriate cellular locale fully intact and in therapeutically relevant concentrations, they must also retain activity upon arrival. The question of residual activity is especially critical when delivery involves exposure to the late endocytic pathway, whose acidic lumenal environment can denature and/or degrade internalized material. ZF5.3 is a compact, stable, rationally designed mini-protein that efficiently escapes intact from late endocytic vesicles, with or without covalently linked protein cargo. Here, using insights from mechanistic studies on the pathway of endosomal escape and classic principles of zinc(II) bioinorganic chemistry, we re-designed the sequence of ZF5.3 to successfully alter the timing (but not the efficiency) of endosomal escape. The new mini-protein we describe, AV5.3, escapes earlier than ZF5.3 along the endocytic pathway with no loss in efficiency, with or without enzyme cargo. More importantly, earlier endosomal escape translates into higher enzymatic activity of a pH-sensitive enzyme upon arrival in the cytosol. Delivery of the pH-sensitive enzyme dihydrofolate reductase (DHFR) with AV5.3 results in substantial catalytic activity in the cytosol, whereas delivery with ZF5.3 does not. The activity of AV5.3-DHFR upon delivery is sufficient to rescue a genetic DHFR deletion in CHO cells. This work provides evidence that programmed trafficking through the endosomal pathway is a viable strategy for the efficient cytosolic delivery of therapeutic proteins. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/615476v2_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1de8a77org.highwire.dtl.DTLVardef@154c401org.highwire.dtl.DTLVardef@105ce8org.highwire.dtl.DTLVardef@43c1f3_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Vazquez Maldonado, A. L., Chen, T., Rodriguez, D., Zoltek, M., Schepartz, A.. 2024-09-28. Earlier endosomal escape improves the catalytic activity of delivered enzyme cargo. https://doi.org/10.1101/2024.09.27.615476

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