CT-159 Precise Measurement of Transduction Efficiency at Single-Cell Resolution for Cell Therapy Development

Abstract

Cell and gene therapies are transformative solutions for a host of inherited and acquired diseases for which existing interventions are ineffective. Many such therapies rely on the introduction of transgenes into host cells using viral or non-viral vectors. Unlike most small molecule drugs, this approach offers long-term benefits and may even serve as a curative solution. The accurate measurement of gene transfer is critical to the development of therapeutic agents and is a key attribute for assessing their safety and efficacy. Yet, conventional methods for measuring gene transfer either report a population average (bulk) or involve time-consuming clonal outgrowth. Here, we demonstrate that single-cell DNA sequencing identifies transduced versus non-transduced cells with exceptional accuracy and precision for populations of up to 10,000 cells. Cells were transduced with lentivirus containing a unique genetic sequence and were then diluted with non-transduced cells to achieve roughly 0, 25, 50, 75, and 100% of positively transduced cells. Five replicates from each concentration were then quantified using Mission Bio's Tapestri Platform, an instrument that leverages droplet microfluidics to enable the targeting and sequencing of DNA in 1,000s of individual cells. The data were analyzed using Tapestri proprietary software. The dilution series showed excellent linearity (R2 greater than 0.99) and precision among replicates (%CV's below 5%) between the expected and observed transduction percentages. For the 5 replicates of non-transduced cells, the false positive rate was below 0.03%. This study showcases the ability of single-cell DNA sequencing to accurately report the percentage of positively transduced cells in a single high-throughput assay without the need for tedious single-cell isolations and clonal outgrowth. Single-cell DNA sequencing offers exciting new capabilities for the development of in vivo and ex vivo cell and gene therapies. By precisely measuring the presence or absence of viral DNA on a per cell basis, researchers can quickly optimize gene transfer protocols during preclinical development, clinical trials and batch release testing during cell product manufacturing.