Biology subjects

Ekman, F. K.

Publications and source records attributed to Ekman, F. K..

3 recordsLinked to original sources

bioRxiv2026

TFU72 is a novel and potent DNA-PKcs inhibitor for enhancing homology-directed repair gene editing

Precise gene editing through homology directed repair (HDR) is one of the most versatile genome editing approaches with broad applications. Achieving high HDR gene editing efficiency is critical to realizing the full potential of this approach. Although many strategies have been explored to enhance HDR editing efficiency, inhibition of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a key component of the non-homologous end joining (NHEJ) pathway remains one of the most effective. Here we describe a novel, highly potent DNA-PKcs small molecule inhibitor, TFU72 which enhances HDR gene editing efficiency remarkably by up to 30-fold in cell lines and human primary cells. We assessed the previously reported genotoxic outcomes associated with DNA-PKcs inhibition such as off-target mutations, chromosomal translocations and large deletions and describe approaches to mitigate these outcomes to safely enhance HDR gene editing efficiency with TFU72. This optimized approach enables broad application of TFU72 for HDR-based precise gene editing applications in both therapeutic and research settings.

molecular biology↗

bioRxiv2025

Engineering hematopoietic stem and progenitor cells to generate red blood cells as viral traps against HIV-1

Canonical HIV-1 entry into target cells depends on binding to CD4 as a primary receptor. Because of this, use of the CD4 receptor as a viral trap (a decoy receptor used to prevent infection of target cells) is a promising strategy for the treatment of HIV-1. One challenge in using CD4 viral traps is maintaining enough of the decoy receptor in circulation to remain effective. Here we present a strategy to produce cell-based CD4 viral traps by engineering hematopoietic stem and progenitor cells (HSPCs) to express the CD4 receptor in red blood cell (RBC) progeny. This takes advantage of the ability of the HSPC to repopulate the blood system for a lifetime, while leveraging the fact that RBCs greatly outnumber any cell targeted for infection. Engineered HSPCs efficiently express CD4 on their cell surface after differentiation to the RBC lineage in vitro. Fusion of CD4 to glycophorin A (GPA) and introduction of a truncated erythropoietin receptor (tEPOR) leads to increased CD4 expression and enrichment of edited cells (CD4-GPA-tEPOR) to levels capable of neutralizing HIV-1 pseudovirus in vitro. In sum, this work presents a potential strategy for the one-time delivery of CD4-RBC viral traps through autologous transplantation of engineered HSPCs.

bioengineering↗

bioRxiv2024

Engineering inducible signaling receptors to enable erythropoietin-free erythropoiesis

Blood transfusion plays a vital role in modern medicine. However, availability is contingent on donated blood, and frequent shortages pose a significant healthcare challenge. Ex vivo manufacturing of red blood cells (RBCs) derived from universal donor O-negative pluripotent stem cells emerges as a solution, yet the high cost of recombinant cytokines required for ex vivo erythroid differentiation remains a major barrier. Erythropoietin (EPO) signaling through the EPO receptor is indispensable to RBC development, and EPO is one of the most expensive components in erythroid-promoting media. Here, we used design-build-test cycles to develop highly optimized small molecule-inducible EPO receptors (iEPORs) which were integrated at a variety of genomic loci using homology-directed repair genome editing. We found that integration of iEPOR at the endogenous EPOR locus in an induced pluripotent stem cell producer line enabled culture with small molecule to yield equivalent erythroid differentiation, transcriptomic changes, and hemoglobin production compared to cells cultured with EPO. Due to the dramatically lower cost of small molecules vs. recombinant cytokines, these efforts eliminate one of the most expensive elements of ex vivo culture media--EPO cytokine. Because dependence on cytokines is a common barrier to ex vivo cell production, these strategies could improve scalable manufacturing of a wide variety of clinically relevant cell types. More broadly, this work showcases how synthetic biology and genome editing may be combined to introduce precisely regulated and tunable behavior into cells, an advancement which will pave the way for increasingly sophisticated cell engineering strategies.

synthetic biology↗