Biology subjects

Lin, R.-Z.

Publications and source records attributed to Lin, R.-Z..

4 recordsLinked to original sources

bioRxiv2024

CRISPR-Enabled Autonomous Transposable Element (CREATE) for RNA-based gene editing and delivery

To address a wide range of genetic diseases, genome editing tools that can achieve targeted delivery of large genes without causing double-stand breaks (DSBs) or requiring DNA templates are necessary. Here, we introduce the CRISPR-Enabled Autonomous Transposable Element (CREATE), a genome editing system that combines the programmability and precision of CRISPR/Cas9 with the RNA-mediated gene insertion capabilities of the human LINE1 (L1) element. CREATE employs a modified L1 mRNA to carry a payload gene, and a Cas9 nickase to facilitate targeted editing by L1-mediated reverse transcription and integration without relying on DSBs or DNA templates. Using the system, a 1.1 kb gene cassette comprising an EF1 promoter and green fluorescent protein (GFP) gene is inserted into several genomic loci of multiple human cell lines. Mechanistic studies reveal that the CREATE system is highly specific with no observed off-target events. Together, these findings establish CREATE as a programmable gene delivery technology solely based on RNA components, enabling large-scale in vivo genome engineering with broad therapeutic potential.

synthetic biology↗

bioRxiv2022

Enhanced intramyocardial vascular cell delivery promotes post-myocardial infarction healing by polarizing pro-regenerative neutrophils

The success of vascular progenitor cell transplantation to treat myocardial infarction (MI) is primarily limited by the low engraftment of delivered cells due to a washout effect during myocardium contraction. A clinically applicable biomaterial to improve cell retention is arguably needed to enable optimization of intramyocardial cell delivery. Here, we developed a novel therapeutic cell delivery method for MI treatment based on a photocrosslinkable gelatin methacryloyl (GelMA) hydrogel. A combination of human vascular progenitor cells (endothelial progenitors and mesenchymal stem cells) with the capacity to form functional vasculatures after transplantation, were injected with a rapid in-situ photopolymerization approach into the infarcted zone of mouse hearts. Our approach significantly improved acute cell retention and achieved a long-term beneficial post-MI cardiac healing, including stabilizing cardiac functions, preserving viable myocardium, and preventing cardiac fibrosis. Furthermore, the engrafted vascular cells polarized recruited bone marrow-derived neutrophils toward a non-inflammatory phenotype via TGF{beta} signaling, establishing a pro-regenerative microenvironment. Depletion of neutrophils canceled the therapeutic benefits produced by cell delivery in the ischemic hearts, indicating that the non-inflammatory, pro-regenerative neutrophils were indispensable mediators of cardiac remodeling. In summary, our novel GelMA hydrogel-based intramyocardial vascular cell delivery approach has the potential to improve the treatment of acute MI.

bioengineering↗

bioRxiv2020

Robust differentiation of human pluripotent stem cells into endothelial cells via temporal modulation of ETV2 with modified mRNA

Human induced pluripotent stem cell (h-iPSC)-derived endothelial cells (h-iECs) have become a valuable tool in regenerative medicine. However, current differentiation protocols remain inefficient and lack reliability. Here, we describe a method for rapid, consistent, and highly efficient generation of h-iECs. The protocol entails the delivery of modified mRNA encoding the transcription factor ETV2 at the intermediate mesodermal stage of differentiation. This approach reproducibly differentiated thirteen diverse h-iPSC lines into h-iECs with exceedingly high efficiency. In contrast, standard differentiation methods that relied on endogenous ETV2 were inefficient and notably inconsistent. Our h-iECs were functionally competent in many respects, including the ability to form perfused vascular networks in vivo. Importantly, timely activation of ETV2 was critical, and bypassing the mesodermal stage produced putative h-iECs with reduced expansion potential and inability to form functional vessels. Our protocol has broad applications and could reliably provide an unlimited number of h-iECs for vascular therapies.

cell biology↗

bioRxiv2018

Role of anterograde motor Kif5b in clathrin-coated vesicle uncoating and clathrin-mediated endocytosis

Kif5b-driven anterograde transport and clathrin-mediated endocytosis (CME) are responsible for opposite intracellular trafficking, contributing to plasma membrane homeostasis. However, whether and how the two trafficking processes coordinate remain unclear. Here, we show that Kif5b directly interacts with clathrin heavy chain (CHC) at a region close to that for uncoating catalyst (Hsc70) and preferentially localizes on large clathrin-coated vesicles (CCVs). Uncoating in vitro is decreased for CCVs from the cortex of kif5b conditional knockout (mutant) mouse and facilitated by adding CHC-binding Kif5b fragments, while cell peripheral distribution of CHC or Hsc70 keeps unaffected by Kif5b depletion. Furthermore, cellular entry of vesicular stomatitis virus that internalized into large CCV is inhibited in cells by Kif5b depletion or introducing a dominant-negative Kif5b fragment. These findings showed a new role of Kif5b in CCV uncoating and CME, indicating Kif5b as a molecular knot connecting anterograde transport to CME.

cell biology↗