Biology subjects

Seow, Y.

Publications and source records attributed to Seow, Y..

3 recordsLinked to original sources

bioRxiv2026

In situ loading of biologics on and tissue redirection of circulating extracellular vesicles with an anchoring nanobody

Extracellular vesicles (EVs) are promising nanocarriers of therapeutic macromolecules, but ex vivo production, cargo loading and quality control constrains clinical translation. Here we demonstrate that NEV3, an EV-anchoring nanobody identified by phage display, binds EVs from multiple sources through interactions involving fibronectin and loads diverse proteins onto EVs, resulting in enhanced intracellular delivery and biological activities in vitro and in vivo. Conjugating NEV3 to target proteins hijacks circulating EVs, prolonging circulation and altering tissue distribution in vivo. When fused with an GFP nanobody or protein A, NEV3 enabled simultaneous capture and loading of circulating GFP or immunoglobulin G (IgG) antibodies on endogenous EVs with enhanced intracellular delivery in vitro and redistribution to liver in vivo. Intravenous protein A-NEV3 significantly reduced circulating IgG and anti-dsDNA antibodies in systemic lupus erythematosus mice with improved survival and reduced pathologies. Intravenous anti-transferrin receptor-protein A-NEV3 redirected circulating EVs to muscle and alleviated muscle pathologies in dystrophin-deficient mdx mice. Our study demonstrates that NEV3 enables in situ loading of protein therapeutics and tissue-specific delivery of circulating EVs, providing a modular strategy for loading cargo onto and redirecting circulating EVs in vivo.

bioengineering↗

bioRxiv2026

MethylAmp One-step isothermal amplification with preservation of DNA methylation patterns

DNA methylation is an important epigenetic modification involved in gene regulation, genome stability, development, and disease. Analysis of low-abundance methylated DNA often requires DNA amplification. Conventional amplification methods, however, do not preserve methylation marks on newly synthesized DNA, potentially compromising downstream methylation analysis. Recently, DNA methyltransferase 1 (DNMT1) was coupled with phi29-mediated whole-genome amplification to preserve DNA methylation for downstream bisulfite sequencing. Targeted amplification of short, predefined genomic loci requires a different amplification strategy. Here, we developed MethylHDA, a one-pot isothermal workflow that integrates primer-directed helicase-dependent amplification (HDA) with DNMT1-mediated maintenance of DNA methylation. Because standard thermophilic HDA is performed at approximately 65 {degrees}C, a temperature incompatible with sustained DNMT1 activity, we established a unified reaction buffer and operating temperature of 42 {degrees}C that supported both HDA and DNMT1. Under these conditions, HDA produced an approximately five-cycle reduction in qPCR threshold cycle (Ct) relative to the unamplified control. Methylation-sensitive restriction enzyme quantitative PCR (MSRE-qPCR) demonstrated that inclusion of DNMT1 increased retention of methylation-dependent restriction protection and that the methylation signal was proportional to the methylated fraction of the input templates. These results demonstrate the feasibility of coupling targeted isothermal DNA amplification with concurrent maintenance of DNA methylation in a single reaction. This approach provides a foundation for methylation-preserving preamplification of short, locus-specific targets for downstream MSRE-qPCR and other targeted methylation assays.

molecular biology↗

bioRxiv2020

Engineering of extracellular vesicles for display of protein biotherapeutics

Extracellular vesicles (EVs) have recently emerged as a highly promising cell-free bio-therapeutics. While a range of engineering strategies have been developed to functionalize the EV surface, current approaches fail to address the limitations associated with endogenous surface display, pertaining to the heterogeneous display of commonly used EV-loading moieties among different EV subpopulations. Here we present a novel engineering platform to display multiple protein therapeutics simultaneously on the EV surface. As proof-of-concept, we screened multiple endogenous display strategies for decorating the EV surface with cytokine binding domains derived from tumor necrosis factor receptor 1 (TNFR1) and interleukin 6 signal transducer (IL6ST), which can act as decoys for the pro-inflammatory cytokines TNF and IL6, respectively. Combining synthetic biology and systematic screening of loading moieties, resulted in a three-component system which increased the display and decoy activity of TNFR1 and IL6ST, respectively. Further, this system allowed for combinatorial functionalization of two different receptors on the same EV surface. These cytokine decoy EVs significantly ameliorated disease phenotypes in three different inflammatory mouse models for systemic inflammation, neuroinflammation, and intestinal inflammation. Importantly, significantly improved in vitro and in vivo efficacy of these engineered EVs was observed when compared directly to clinically approved biologics targeting the IL6 and TNF pathways.

bioengineering↗