Salma, J. | bioRxiv Science

bioRxiv2026

A TLS11a-decorated ionizable lipid nanoparticle platform and a multilevel-validated CRISPR LDLR-knockout HepG2 model for hepatocyte-preferential mRNA delivery

Ionizable lipid nanoparticles (LNPs) are widely used for delivery of CRISPR/Cas9 payloads to hepatocytes, but conventional hepatic uptake is strongly influenced by adsorption of apolipoprotein E and subsequent low-density lipoprotein receptor (LDLR)-mediated internalization. This dependence may limit specificity and reduce efficacy in LDLR-deficient settings. Here, we designed an aptamer-functionalized LNP platform to enable hepatocyte-selective genome editing through an LDLR-independent route and validated its performance using a genetically defined LDLR-knockout HepG2 model. Ionizable LNPs co-encapsulating Cas9 mRNA and an LDLR-targeting guide RNA were surface-decorated with the hepatocellular carcinoma-targeting TLS11a aptamer using thiol-maleimide chemistry. Comprehensive physicochemical analysis using cryo-electron microscopy, dynamic light scattering, pKa titration, UV and circular dichroism spectroscopy, X-ray photoelectron spectroscopy, and molecular beacon assays confirmed uniform nanoparticles of approximately 105 nm, preserved mRNA integrity, retained endosomal charge-switching behavior with a pKa of approximately 6.3 to 6.5, and maintained correctly folded surface-displayed TLS11a. TLS11a decoration increased Cas9 mRNA delivery to HepG2 cells from 39% to 79% Cy5-positive cells, while reducing uptake in receptor-low control cells, supporting aptamer-associated and cell-preferential delivery. In parallel, CRISPR/Cas9-mediated deletion of LDLR exon 2 generated a validated LDLR-deficient HepG2 line, confirmed at genomic, transcript, and protein levels. LDLR loss reduced LDL binding and uptake by approximately 85%, while transferrin uptake was preserved, indicating selective impairment of LDLR-dependent endocytosis. Cholesterol depletion activated the SCAP-SREBP-2 pathway and induced cholesterol biosynthesis genes. Together, these findings establish a modular aptamer-guided LNP system for targeted genome-editing delivery and a validated LDLR-null hepatocyte model for studying LDLR-dependent biology and disease.

bioengineering↗

bioRxiv2021

Tropism of SARS-CoV-2 for Developing Human Cortical Astrocytes

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) readily infects a variety of cell types impacting the function of vital organ systems, with particularly severe impact on respiratory function. It proves fatal for one percent of those infected. Neurological symptoms, which range in severity, accompany a significant proportion of COVID-19 cases, indicating a potential vulnerability of neural cell types. To assess whether human cortical cells can be directly infected by SARS-CoV-2, we utilized primary human cortical tissue and stem cell-derived cortical organoids. We find significant and predominant infection in cortical astrocytes in both primary and organoid cultures, with minimal infection of other cortical populations. Infected astrocytes had a corresponding increase in reactivity characteristics, growth factor signaling, and cellular stress. Although human cortical cells, including astrocytes, have minimal ACE2 expression, we find high levels of alternative coronavirus receptors in infected astrocytes, including DPP4 and CD147. Inhibition of DPP4 reduced infection and decreased expression of the cell stress marker, ARCN1. We find tropism of SARS-CoV-2 for human astrocytes mediated by DPP4, resulting in reactive gliosis-type injury.

neuroscience↗