Biology subjects

Schissel, C. K.

Publications and source records attributed to Schissel, C. K..

7 recordsLinked to original sources

bioRxiv2026

High-throughput discovery of arginine-depleted peptides enables effective antisense delivery for Duchenne muscular dystrophy

Phosphorodiamidate morpholino oligomers (PMOs) are approved exon-skipping antisense therapeutics for Duchenne muscular dystrophy (DMD), but their clinical utility is limited by poor uptake in muscle tissue, necessitating frequent high-dose administration. Cell-penetrating peptides (CPPs) can enhance intracellular delivery of PMOs, yet conventional arginine-rich CPPs often cause dose-limiting toxicity, including renal damage, which hinders their clinical translation. To address this challenge, we developed a high-throughput, charge-based chromatographic enrichment platform capable of screening over 15,000 synthetic peptides, including sequences with noncanonical (abiotic) amino acids. This approach enabled de novo discovery of arginine-depleted CPPs with improved delivery profiles. Four lead candidates demonstrated efficient nuclear PMO delivery with ~10-fold lower in vitro toxicity compared to standard CPPs such as penetratin. The top-performing peptide, CXP1, showed robust splice-switching activity and favorable tolerability in both cellular and animal models. In dystrophic mdx mice, CXP1-PMO conjugates achieved greater exon skipping compared to PMOs conjugated to R6G at equivalent doses. Tissue levels of CXP1-PMO correlated with exon-skipping efficacy, establishing a clear pharmacokinetic-pharmacodynamic relationship. These findings highlight a mechanistically novel and translationally relevant discovery strategy, demonstrating the potential of high-throughput platforms to generate more effective CPP-based delivery vehicles for antisense therapeutics in DMD and related neuromuscular disorders. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=44 SRC="FIGDIR/small/730741v1_ufig1.gif" ALT="Figure 1"> View larger version (13K): org.highwire.dtl.DTLVardef@84b7f2org.highwire.dtl.DTLVardef@1487850org.highwire.dtl.DTLVardef@d2e263org.highwire.dtl.DTLVardef@10ce371_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

Monitoring monomer-specific acyl-tRNA levels in cells with PARTI

We describe a new assay that reports directly on the acylation state of a user-chosen tRNA in cells. We call this assay 3-Prime Adenosine-Retaining Aminoacyl-tRNA Isolation (PARTI). It relies on high-resolution mass spectrometry identification of the acyl-adenosine species released upon RNase A cleavage of isolated cellular tRNA. Here we develop the PARTI workflow and apply it to understand three recent observations related to the cellular incorporation of non--amino acid monomers into protein: (1) the origins of the apparent selectivity of translation with respect to {beta}2-hydroxy acid enantiomers; (2) the activity of PylRS variants for benzyl derivatives of malonic acid; and (3) the apparent inability of N-Me amino acids to function as ribosome substrates in living cells. Using the PARTI assay, we also provide direct evidence for the cellular production of 2,3-diacylated tRNA in certain cases. The ease and simplicity of the PARTI workflow should benefit ongoing efforts to study and improve the cellular incorporation of non--amino acid monomers into proteins. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/633079v2_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@f3b8a0org.highwire.dtl.DTLVardef@de400dorg.highwire.dtl.DTLVardef@13f17faorg.highwire.dtl.DTLVardef@13f2c0f_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Backbone extension acyl rearrangements enable cellular synthesis of proteins with internal β2-peptide linkages

Proteins and polypeptides containing extended backbone monomers embody highly desirable structures and functions, but they cannot yet be biosynthesized in cells. There are two challenges at work. First is the ribosome, whose ability to promote rapid bond-forming reactions to and from anything other than an -amino acid or -hydroxy acid is unknown. The second challenge is the absence of orthogonal enzymes that acylate tRNA with extended backbone monomers. Here we describe a general approach to the programmed cellular synthesis of proteins containing extended backbone monomers that circumvents both of these challenges. Rather than relying on direct and uncharacterized reactions of non--amino acid monomers within the ribosomal PTC, we develop a proximity-guided intramolecular rearrangement that effectively edits the protein backbone post-translationally. The method relies on the ability of PylRS-like aminoacyl-tRNA synthetase enzymes to accept diverse -hydroxy acid monomers, including those whose side chains contain masked nucleophiles. Introduction of such an -hydroxy acid monomer into a protein translated in vivo, followed by nucleophile unmasking, sets up a thermodynamically favored and quantitative intramolecular Backbone Extension Acyl Rearrangement (BEAR) reaction that edits the protein backbone to install an extended backbone monomer. In the examples described here, the intramolecular rearrangement converts an -peptide backbone directly into a {beta}-backbone. As far as we know, this report represents the first example in which a much-desired expanded backbone {beta}-amino acid linkage has been introduced site-selectively into a protein in a cell.

synthetic biology↗

bioRxiv2022

In-cell penetration selection-mass spectrometry produces noncanonical peptides for antisense delivery

Peptide-mediated delivery of macromolecules in cells has significant potential therapeutic benefits, but no therapy employing cell-penetrating peptides (CPPs) has reached the market after 30 years of investigation due to challenges in the discovery of new, more efficient sequences. We developed a method for in-cell penetration selection-mass spectrometry (in-cell PS-MS) to discover peptides from a synthetic library capable of delivering macromolecule cargo to the cytosol. This method was inspired by recent in vivo selection approaches for cell-surface screening, with an added spatial dimension resulting from subcellular fractionation. A representative peptide discovered in the cytosolic extract, Pep1a, is nearly 100-fold more active toward antisense phosphorodiamidate morpholino oligomer (PMO) delivery compared to a sequence identified from a whole cell extract, which includes endosomes. Pep1a is composed of D-amino acids and two non--amino acids. Pulse-chase and microscopy experiments revealed that while the PMO-Pep1a conjugate is likely taken up by endosomes, it can escape to localize to the nucleus. In-cell PS-MS introduces a means to empirically discover unnatural synthetic peptides for subcellular delivery of therapeutically relevant cargo.

bioengineering↗

bioRxiv2021

Cell-Penetrating D-Peptides Retain Antisense Morpholino Oligomer Delivery Activity

Cell-penetrating peptides (CPPs) can cross the cell membrane to enter the cytosol and deliver otherwise non-penetrant macromolecules such as proteins and oligonucleotides. For example, recent clinical trials have shown that a CPP attached to phosphorodiamidate morpholino oligomers (PMO) resulted in higher muscle concentration, increased exon-skipping and dystrophin production relative to another study of the PMO alone in patients of Duchenne muscular dystrophy. Therefore, effective design and study of CPPs could help enhance therapies for difficult-to-treat diseases. So far, the study of CPPs for PMO delivery has been restricted to predominantly canonical L-peptides. We hypothesized that mirror-image D-peptides could have similar PMO delivery activity as well as enhanced proteolytic stability, facilitating their characterization and quantification from biological milieu. We found that several enantiomeric peptide sequences could deliver a PMO-biotin cargo with similar activities, while remaining stable against serum proteolysis. The biotin label allowed for affinity capture of fully intact PMO-peptide conjugates from whole cell and cytosolic lysates. By profiling a mixture of these constructs in cells, we determined their relative intracellular concentrations. When combined with PMO activity, these concentrations provide a new metric for delivery efficiency which may be useful for determining which peptide sequence to pursue in further pre-clinical studies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=92 SRC="FIGDIR/small/462617v1_ufig1.gif" ALT="Figure 1"> View larger version (12K): org.highwire.dtl.DTLVardef@887d8borg.highwire.dtl.DTLVardef@ea664aorg.highwire.dtl.DTLVardef@e1b1e1org.highwire.dtl.DTLVardef@1652d47_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2020

Erythrocyte-targeted immunomodulatory antigens enabled by in vivo selection of D-peptides

Targeting of antigens to erythrocytes can be used to selectively mitigate their immunogenicity, but the methods to equip a variety of cargoes with erythrocyte-targeting properties are limited. Here we identified a D-peptide that targets murine erythrocytes and decreases anti-drug antibody responses when conjugated to the protective antigen from Bacillus anthracis, a protein of therapeutic interest. The D-peptide likewise decreases inflammatory anti-ovalbumin (OVA) CD8+ T cell responses when attached to a peptide antigen derived from OVA. To discover this targeting ligand, we leveraged mass spectrometry to decode a randomized D-peptide library selected in mice, extending the application of synthetic libraries to in vivo affinity selections.

bioengineering↗

bioRxiv2020

Interpretable Deep Learning for De Novo Design of Cell-Penetrating Abiotic Polymers

There are more amino acid permutations within a 40-residue sequence than atoms on Earth. This vast chemical search space hinders the use of human learning to design functional polymers. Here we couple supervised and unsupervised deep learning with high-throughput experimentation to drive the design of high-activity, novel sequences reaching 10 kDa that deliver antisense oligonucleotides to the nucleus of cells. The models, in which natural and unnatural residues are represented as topological fingerprints, decipher and visualize sequence-activity predictions. The new variants boost antisense activity by 50-fold, are effective in animals, are nontoxic, and can also deliver proteins into the cytosol. Machine learning can discover functional polymers that enhance cellular uptake of biotherapeutics, with significant implications toward developing therapies for currently untreatable diseases. One sentence summaryDeep learning generates de novo large functional abiotic polymers that deliver antisense oligonucleotides to the nucleus.

bioinformatics↗