RT-qPCR and split-luciferase assays enable batch standardization and analysis of engineered virus-like particle transduction.

Recently developed engineered virus-like particles (eVLPs) have emerged as a promising delivery vehicle for ribonucleoprotein gene editing complexes. Variability in eVLP batch production may, however, hinder reproducibility and standardization across pre-clinical investigations designed to characterize and optimize this platform. Ultimately, stringent production, purification, and quantification processes will be required for this technology to reach the clinic. In this study, we developed two titration methods for base editor (BE)-eVLPs: a spacer-agnostic quantitative reverse-transcription PCR (RT-qPCR) assay to quantify the copy number of sgRNA molecules, and a NanoBiT luciferase-based approach to estimate vesicular stomatitis virus envelope glycoprotein (VSV-G) abundance, per μL of BE-eVLP preparation. We further engineered an LgBiT-expressing reporter cell line to monitor BE-eVLP transduction kinetics in real time. Our findings reveal that both RT-qPCR and HiBiT-based quantification enable effective batch-to-batch standardization of BE-eVLP preparations. Further, the LgBiT-expressing reporter cell line was effective in real-time monitoring of transduction kinetics. BE-eVLP transduction was proven dependent on endosomal acidification and was constrained by cellular endocytic capacity. Paired with accurate quantification of BE-eVLP preparations and using HiBiT-tagged virus-surface glycoproteins, the LgBiT reporter cell line can facilitate comparison of transduction and levels of editing achieved across emergent eVLP platforms and pseudotypes.