Lipid nanoparticle formulation and characterization
Formulated nucleic acid lipid nanoparticles by microfluidics and T-mixer on NanoAssemblr, from 10 microgram to 10 milligram scale, and tested organ-selective and ligand-targeted particles produced by partner teams. Characterization covered dynamic light scattering for size, polydispersity index, and zeta potential, encapsulation efficiency by RiboGreen, cryogenic electron microscopy, and mRNA integrity by TBE-urea gel, agarose gel, and Bioanalyzer. Buffer exchange by PD-10, Amicon ultrafiltration, and dialysis, with small-scale sterile filtration and freeze-thaw and storage stability screens.
Delivery biology and trafficking
Dissected the steps between particle and payload: time-course uptake kinetics, colocalization with endosomal and lysosomal markers, endosomal escape readouts, and quantitative receptor density measurement by flow cytometry to explain why targeted particles worked in some cell types and not others. Worked across primary and immortalized cells, with amplicon sequencing for editing outcomes.
Viral vector engineering
Built lentiviral packaging and producer cell lines in adherent and suspension formats, mostly second generation with some third generation design, then screened and banked producer and transduced lines. Produced and characterized vector by ultracentrifugation, p24, and physical and functional titer, measured vector copy number by droplet digital PCR, and ran next generation sequencing for integration site analysis. Also designed and quality-controlled adeno-associated virus constructs.
Assay development and qualification
Developed droplet digital PCR assays for biodistribution and tropism, improving signal to noise 100-fold on one program and qualifying assays against predefined sensitivity, specificity, and signal to noise criteria that outperformed next generation sequencing readouts by 10 to 100 fold. Wrote protocols with acceptance criteria, characterized assay performance against them, and devised encapsulation assays for cargoes with no established method.
Molecular cloning and construct design
Did most of my own cloning since 2011. Gibson and NEBuilder assembly, Gateway cloning, restriction and ligation, and site-directed mutagenesis, including large and difficult constructs built in recombination-deficient strains. Cloned pooled 2-ICD and 3-ICD CAR-T libraries combining 12 to 15 intracellular domains, single guide RNA libraries, and scFv constructs, in manual 96-well format. Verified by Sanger sequencing, whole plasmid sequencing, and next generation sequencing, with analysis in SnapGene, Benchling, and custom scripts.
Cell biology, flow cytometry, and imaging
Cultured and engineered immortalized and primary cells, including immune cells, with nucleofection, lipid transfection, and lipid nanoparticle treatment. Flow cytometry for receptor density, viability, activation, proliferation, and exhaustion, analyzed in FlowJo, plus FACS sorting. Imaging on Olympus FLUOVIEW FV3000, Zeiss LSM 510 and 710 confocals, DeltaVision Elite, Incucyte, Thermo Fisher CX7 high-content, and IVIS.
Genomics and functional screening
Ran CRISPR screens and lentiviral short hairpin RNA knockdown in immune cells, designed siRNA sequences with computational biologists and confirmed knockdown by Western blot and qRT-PCR, and prepared single-cell RNA sequencing and whole genome sequencing libraries with analysis alongside bioinformatics colleagues. Designed barcoded sequences for high-throughput screening and mined DepMap and TCGA for target prioritization.
Collaboration and program work
Managed contract research and manufacturing partners for plasmids, primers, and synthetic gene blocks, and for in vivo dosing studies where I set groups, intervals, timepoints, and readouts. Presented to leadership and governance reviews, contributed to target selection, lead optimization, and candidate nomination decisions, and set priorities, assigned work, and contributed to performance reviews for junior scientists. Electronic lab notebooks in Benchling and Arxspan.