Biology subjects

Zoltek, M.

Publications and source records attributed to Zoltek, M..

5 recordsLinked to original sources

bioRxiv2024

Packaged delivery of CRISPR-Cas9 ribonucleoproteins accelerates genome editing

Effective genome editing requires a sufficient dose of CRISPR-Cas9 ribonucleoproteins (RNPs) to enter the target cell while minimizing immune responses, off-target editing and cytotoxicity. Clinical use of Cas9 RNPs currently entails electroporation into cells ex vivo, but no systematic comparison of this method to packaged RNP delivery has been made. Here we compared two delivery strategies, electroporation and enveloped delivery vehicles (EDVs), to investigate the Cas9 dosage requirements for genome editing. Using fluorescence correlation spectroscopy (FCS), we determined that >1300 Cas9 RNPs per nucleus are typically required for productive genome editing. EDV-mediated editing was >30-fold more efficient than electroporation, and editing occurs at least two-fold faster for EDV delivery at comparable total Cas9 RNP doses. We hypothesize that differences in efficacy between these methods result in part from the increased duration of RNP nuclear residence resulting from EDV delivery. Our results directly compare RNP delivery strategies, showing that packaged delivery could dramatically reduce the amount of CRISPR-Cas9 RNPs required for experimental or clinical genome editing. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/619117v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@11a1f94org.highwire.dtl.DTLVardef@f3130org.highwire.dtl.DTLVardef@1666938org.highwire.dtl.DTLVardef@85fd13_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Earlier endosomal escape improves the catalytic activity of delivered enzyme cargo

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

biochemistry↗

bioRxiv2024

Requirements for efficient endosomal escape by designed mini-proteins

ZF5.3 is a compact, rationally designed mini-protein that escapes efficiently from the endosomes of multiple cell types. Despite its small size (27 amino acids), ZF5.3 can be isolated intact from the cytosol of treated cells and guides multiple classes of proteins into the cytosol and/or nucleus. In the best cases, delivery efficiencies reach or exceed 50% to establish nuclear or cytosolic concentrations of 500 nM or higher. But other than the requirement for unfoldable cargo and an intact HOPS complex, there is little known about how ZF5.3 traverses the limiting endocytic membrane. Here we delineate the attributes of ZF5.3 that enable efficient endosomal escape. We confirm that ZF5.3 is stable at pH values between 5.5 and 7.5, with no evidence of unfolding even at temperatures as high as 95 {degrees}C. The high-resolution NMR structure of ZF5.3 at pH 5.5, also reported here, shows a canonical p zinc-finger fold with the penta-arg motif integrated seamlessly into the C-terminal -helix. At lower pH, ZF5.3 unfolds cooperatively as judged by both circular dichroism and high-resolution NMR. Unfolding occurs upon protonation of a single Zn(II)-binding His side chain whose pKa corresponds almost exactly to that of the late endosomal lumen. pH-induced unfolding is essential for endosomal escape, as a ZF5.3 analog that remains folded at pH 4.5 fails to efficiently reach the cytosol, despite high overall uptake. Finally, using reconstituted liposomes, we identify a high-affinity interaction of ZF5.3 with a specific lipid-BMP-that is selectively enriched in the inner leaflet of late endosomal membranes. This interaction is 10-fold stronger at low pH than neutral pH, providing a molecular picture for why escape occurs preferentially and in a HOPS-dependent manner from late endosomal compartments. The requirements for programmed endosomal escape identified here should aid and inform the design of proteins, peptidomimetics, and other macromolecules that reach cytosolic or nuclear targets intact and at therapeutically relevant concentrations.

biochemistry↗

bioRxiv2023

Design rules for efficient endosomal escape

The inefficient translocation of proteins across biological membranes limits their application as therapeutic compounds and research tools. In most cases, translocation involves two steps: uptake into the endocytic pathway and endosomal escape. Certain charged or amphiphilic molecules promote protein uptake but few enable efficient endosomal escape. One exception is ZF5.3, a mini-protein that exploits natural endosomal maturation machinery to translocate across endosomal membranes. Although certain ZF5.3-protein conjugates are delivered efficiently into the cytosol or nucleus, overall delivery efficiency varies widely with no obvious design rules. Here we evaluate the role of protein size and thermal stability in the ability to efficiently escape endosomes when attached to ZF5.3. Using fluorescence correlation spectroscopy, a singlemolecule technique that provides a precise measure of intra-cytosolic protein concentration, we demonstrate that delivery efficiency depends on both size and the ease with which a protein unfolds. Regardless of size and pI, low-Tm cargos of ZF5.3 (including intrinsically disordered domains) bias its endosomal escape route toward a high-efficiency pathway that requires the homotypic fusion and protein sorting (HOPS) complex. Small protein domains are delivered with moderate efficiency through the same HOPS portal even if the Tm is high. These findings imply a novel protein- and/or lipid-dependent pathway out of endosomes that is exploited by ZF5.3 and provide clear guidance for the selection or design of optimally deliverable therapeutic cargo. Significance StatementThe results described in this paper provide new insights into how protein delivery works and how it can be best utilized in the future. Although intracellular protein delivery has been studied for decades, this paper describes the first interrogation of why certain protein cargos are privileged for efficient endosomal escape. These results represent a fundamental advance in the long-awaited goal of efficient protein delivery and provide design rules to overcome one of the most significant challenges for the future of biotechnology.

cell biology↗

bioRxiv2022

Dose-dependent nuclear delivery and transcriptional repression with a cell-penetrant MeCP2

Methyl-CpG-binding-protein 2 (MeCP2) is a nuclear protein expressed in all cell types, especially neurons1. Mutations in the MECP2 gene cause Rett syndrome (RTT), an incurable neurological disorder that disproportionately affects young girls2. Strategies to restore MeCP2 expression phenotypically reverse RTT-like symptoms in male and female MeCP2-deficient mice3-5, suggesting that direct nuclear delivery of functional MeCP2 could restore MeCP2 activity. We report that ZF-tMeCP2, a conjugate of MeCP2({Delta}aa13-71, 313-484)6 and the cell-permeant mini-protein ZF5.37-11, both binds DNA in a methylation-dependent manner and reaches the nucleus of model cell lines intact at concentrations above 700 nM. When delivered to live cells, ZF-tMeCP2 engages the NCoR/SMRT co-repressor complex and selectively represses transcription from methylated promoters. Efficient nuclear delivery of ZF-tMeCP2 relies on a unique endosomal escape portal provided by HOPS-dependent endosomal fusion. The Tat conjugate of MeCP2 (Tat-tMeCP2), evaluated for comparison, is degraded within the nucleus, is not selective for methylated promoters, and trafficks in a HOPS-independent manner. These results support the feasibility of a HOPS-dependent portal for delivering functional macromolecules to the cell interior using the cell-penetrant mini-protein ZF5.3. Such a strategy could broaden the impact of multiple families of protein-derived therapeutics.

biochemistry↗