Biology subjects

Luna, S. E.

Publications and source records attributed to Luna, S. E..

4 recordsLinked to original sources

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↗

bioRxiv2024

Combining Cell-Intrinsic and -Extrinsic Resistance to HIV-1 By Engineering Hematopoietic Stem Cells for CCR5 Knockout and B Cell Secretion of Therapeutic Antibodies

Autologous transplantation of CCR5 null hematopoietic stem and progenitor cells (HSPCs) is the only known cure for HIV-1 infection. However, this treatment is limited because of the rarity of CCR5-null matched donors, the morbidities associated with allogeneic transplantation, and the prevalence of HIV-1 strains resistant to CCR5 knockout (KO) alone. Here, we propose a one-time therapy through autologous transplantation of HSPCs genetically engineered ex vivo to produce both CCR5 KO cells and long-term secretion of potent HIV-1 inhibiting antibodies from B cell progeny. CRISPR-Cas9-engineered HSPCs maintain engraftment capacity and multi-lineage potential in vivo and can be engineered to express multiple antibodies simultaneously. Human B cells engineered to express each antibody secrete neutralizing concentrations capable of inhibiting HIV-1 pseudovirus infection in vitro. This work lays the groundwork for a potential one-time functional cure for HIV-1 through combining the long-term delivery of therapeutic antibodies against HIV-1 and the known efficacy of CCR5 KO HSPC transplantation.

bioengineering↗

bioRxiv2023

Using human genetics to develop strategies to increase erythropoietic output from genome-edited hematopoietic stem and progenitor cells

Human genetic polymorphisms result in a diversity of phenotypes. Some sequences are pathologic and lead to monogenic diseases, while others may confer beneficial traits. Genome editing is a powerful tool to recreate genotypes found in the population, including the ability to correct pathologic mutations. One of the best characterized naturally occurring mutations causing congenital erythrocytosis arises from a truncation in the erythropoietin receptor (tEPOR) which can result in non-pathogenic hyper-production of red blood cells (RBCs). Using the precision of CRISPR/Cas9 genome editing, we have recreated tEPOR and studied the effect of variations of the genotype on RBC development. We then combined tEPOR with a correction strategy developed for {beta}-thalassemia and demonstrated that coupling the two genome editing events gave RBCs a significant selective advantage. This demonstrates the potential of combining human genetics with the precision of genome editing to enable safer and more effective genome editing therapies for patients with serious genetic diseases.

genetics↗