Biology subjects

Lawanprasert, A.

Publications and source records attributed to Lawanprasert, A..

4 recordsLinked to original sources

bioRxiv2026

LNP-mediated BCL11A Editing Corrects Sickling Phenotypes and Preserves HSPC Fitness Compared to Electroporation

Hemoglobinopathies, including sickle cell disease (SCD) and thalassemia syndromes, affect millions of individuals worldwide who have limited access to curative therapies. Autologous hematopoietic stem cell transplant following ex vivo CRISPR editing of the BCL11A erythroid enhancer reactivates fetal hemoglobin (HbF) and achieves an effective cure, but the resource constraints of clinically approved procedures for editing by electroporation (EP) severely limit widespread implementation. We directly compared the functional outcomes of EP delivery of Cas9 ribonucleoprotein with lipid nanoparticle (LNP) delivery of Cas9 mRNA in primary human HSPCs obtained from healthy HbAA donors and from patients with SCD. While higher editing rates are achieved with EP, LNP-treated HSPCs exhibited greater viability and cell yields that persisted throughout a multi-stage in vitro erythroid differentiation protocol. By day 20, the yield of mature red blood cells (CD71lowCD235ahigh) was lowest in the EP cohorts. Across treatment groups, we observed HbF induction proportional to indel frequency. LNP editing of SCD patient-derived HSPCs as low as 25% modified alleles still caused HbF production and reduced the propensity for sickling of in vitro differentiated RBCs. These findings highlight the critical trade-offs among manufacturing ease, delivery-associated toxicity, and functional performance across two modalities of therapeutic genome editing for hemoglobinopathies.

bioengineering↗

bioRxiv2026

A lipid nanoparticle platform for high yield CRISPR-targeted homology directed repair enables fully non-viral CAR T cell generation

CRISPR-mediated homology directed repair (HDR) enables targeted CAR integration with improved fitness and therapeutic potential of CAR T cells. However, current methods for generating HDR-engineered CAR T cells rely on viral transduction or electroporation, approaches that limit global implementation and constrain patient access due to their cost, toxicity, and requirement for centralized manufacturing. Through a screen of ionizable lipids, we identified LNP systems that enable CRISPR-mediated gene knock-in (KI) in primary human T cells and are amenable to hand mixing by ethanol injection as a research tool or machine formulation for larger scale manufacturing. Modifying the linear dsDNA HDR template with truncated Cas9 target sequences (tCTS) enhanced HDR rates across multiple LNP systems. We optimized two LNP formulations capable of HDR-mediated KI of a large 4kB CD19 CAR-EGFR HDR template into the TRAC locus with rates of [≥]8% and >10x improved edited cell yields compared to electroporation. We demonstrate that LNP-generated CAR T cells exhibited similar growth kinetics, activation states, differentiation states, and killing capacity compared to electroporation-generated CAR T cells. Our LNP platform components are fully disclosed, commercially sourced, and enable efficient fully non-viral CRISPR-HDR cell engineering across diverse applications.

bioengineering↗

bioRxiv2025

CRISPR-Cas13d-mediated targeting of a context-specific essential gene enables selective elimination of uveal melanoma

Uveal melanoma, the most common eye cancer in adults, remains limited to surgical intervention and chemotherapy, with a dismal survival rate that has not improved in over 50 years. To address this therapeutic impasse, we systematically analyzed public gene expression, RNAi, and CRISPR knockout datasets and identified RASGRP3 as an essential gene specifically for uveal melanoma. RasGRP3 is uniquely overexpressed and essential for survival in uveal melanoma cells, but dispensable in healthy cells. RasGRP3 remains "undruggable" due to its intracellular localization and lack of targetable binding pockets. To overcome this, we developed a CRISPR-Cas13d RNA-targeting therapeutic that specifically knocks down RasGRP3 mRNA. This Cas13d-based therapeutic mediates selective uveal melanoma killing through two synergistic mechanisms: (i) direct silencing of the essential RasGRP3 transcript, and (ii) collateral RNA degradation triggered by the cleavage of overexpressed RasGRP3. When delivered via optimized lipid nanoparticles encoding Cas13d mRNA and guide RNA, this strategy eliminated >97% of uveal melanoma cells while sparing healthy cells, including retinal pigment epithelial cells. This approach outperformed conventional Cas9 and siRNA methods in potency without inducing permanent genomic alterations. Our findings establish a RNA-targeting therapeutic for uveal melanoma and a framework for Cas13d-based interventions against broad "undruggable" cancers.

synthetic biology↗

bioRxiv2024

Heat Stable and Intrinsically Sterile Liquid Protein Formulations

Over 80% of biologic drugs, and 90% of vaccines, require temperature-controlled conditions throughout the supply chain to minimize thermal inactivation and contamination. This cold chain is costly, requires stringent oversight, and is impractical in remote environments. Here, we report chemical dispersants that non-covalently solvate proteins within fluorous liquids to alter their thermodynamic equilibrium and reduce conformational flexibility. This generates non-aqueous, fluorine-based liquid protein formulations that biochemically rigidify protein structure to yield thermally stable biologics at extreme temperatures (up to 90{degrees}C). These non-aqueous formulations are impervious to contamination by microorganismal pathogens, degradative enzymes, and environmental impurities, and display comparable pre-clinical serum half-life and safety profiles to standard saline protein samples. As a result, we deliver a fluorochemical formulation paradigm that may limit the need for cold chain logistics of protein reagents and biopharmaceuticals.

biophysics↗