Kwon, E. J. | Search bioRxiv

bioRxiv2022

An Activity-Based Nanosensor for Minimally-Invasive Measurement of Protease Activity in Traumatic Brain Injury

Current screening and diagnostic tools for traumatic brain injury (TBI) have limitations in sensitivity and prognostication. Aberrant protease activity is a central process that drives disease progression in TBI and is associated with worsened prognosis; thus direct measurements of protease activity could provide more diagnostic information. In this study, we engineered a nanosensor that releases a measurable signal into the blood and urine in response to activity from the TBI-associated protease calpain. Readouts from our nanosensor were designed to be compatible with ELISA and lateral flow assays, clinically-relevant assay modalities. In a mouse model of TBI, we demonstrated greater sensitivity of the nanosensor with the addition of targeting ligands to hyaluronic acid. In evaluation of mice with mild or severe injuries, our nanosensor identified mild TBI with a higher sensitivity than the clinical biomarker GFAP. This nanosensor technology allows for measurement of TBI-associated proteases without the need to directly access brain tissue, and has the potential to complement existing TBI diagnostic tools.

bioengineering↗

bioRxiv2022

Extensive in vitro and in vivo protein translation via in situ circularized RNAs

RNAs are a powerful therapeutic class. However their inherent transience impacts their activity both as an interacting moiety as well as a template. Circularization of RNA has been demonstrated as a means to improve persistence, however simple and scalable approaches to achieve this are lacking. Utilizing autocatalytic RNA circularization, here we engineer in situ circularized RNAs (icRNAs). This approach enables icRNA delivery as simple linear RNA that is circularized upon delivery into the cell, thus making them compatible with routine synthesis, purification, and delivery formulations. We confirmed extensive protein translation from icRNAs both in vitro and in vivo and explored their utility in three contexts: first, we delivered the SARS-CoV-2 Omicron spike protein in vivo as icRNAs and showed corresponding induction of humoral immune responses; second, we demonstrated robust genome targeting via zinc finger nucleases delivered as icRNAs; and third, to enable compatibility between persistence of expression and immunogenicity, we developed a novel long range multiplexed (LORAX) protein engineering methodology to screen progressively deimmunized Cas9 proteins, and demonstrated efficient genome and epigenome targeting via their delivery as icRNAs. We anticipate this highly simple and scalable icRNA methodology could have broad utility in basic science and therapeutic applications.

bioengineering↗