Abstract
The ability to deliver transgenes into the human genome using viral vectors is a major enabler of the gene-modified cell therapy field. However, the control of viral transduction is difficult and can lead to product heterogeneity, impacting efficacy and safety, as well as increasing the risk of batch failure during manufacturing. To address this, we generated a novel analytical method to measure vector copy distribution at the single-cell level in a gene-modified, lentiviral-based immunotherapy model. As the limited amount of genomic DNA in a single cell hinders reliable quantification, we implemented a preamplification strategy on selected lentiviral and human gene targets in isolated live single cells, followed by quantification of amplified material by droplet digital PCR. Using a bespoke probability framework based on Bayesian statistics, we show that we can estimate vector copy number (VCN) integers with maximum likelihood scores. Notably, single-cell data are consistent with population analysis and also provide an overall measurement of transduction efficiency by discriminating transduced (VCN ≥ 1) from nontransduced (VCN = 0) cells. The ability to characterize cell-to-cell variability provides a powerful high-resolution approach for product characterization, which could ultimately allow improved control over product quality and safety.
Highlights
Gene-modified cell therapies have the potential to circumvent pathological conditions caused by genetic aberrations by introducing exogenous therapeutic transgenes into host cells
To measure the vector copy number (VCN) of the transduced cells at the population VCN (pVCN) level, we designed three hydrolysis-probe assays targeting RRE (VG1), WPRE (VG2), and the ZsGreen transgene (VG3) vector sequences. These were used in combination with two commercially available human copy number reference gene (RG) assays for ribonuclease P RNA component H1 (RPPH1; RG1) and telomerase reverse transcriptase (TERT; RG2) to generate six unique combinations of duplex droplet digital PCR (ddPCR) reactions (Figures 1B and S1)
Tightly controlled processes are required to ensure these products meet rigorous standards for quality and safety. This is achieved during development and manufacturing through analytical characterization, which is pivotal for monitoring product specifications and critical quality attributes
Summary
Gene-modified cell therapies have the potential to circumvent pathological conditions caused by genetic aberrations by introducing exogenous therapeutic transgenes into host cells. Unlike standard treatments using small-molecule drugs or biopharmaceuticals, which are designed to prevent or manage disease progression, cell and gene therapies often have long-lasting curative outcomes This creates a new way to control disease and has fueled a rapidly growing and evolving field. The ease with which their genome can be engineered and the relatively large cargo (up to 5 kb) they can accommodate have allowed their extensive use in more than 70% of current clinical trials.[4] Vectors belonging to the retroviridae family, such as retroviruses and lentiviruses, can stably integrate into the host genome, providing potential long-term therapeutic benefits These advantages are tempered by the intrinsic risk of insertional mutagenesis, which may occur when viral integration impairs the functionality of proto-oncogenes.[5,6,7,8,9] To address concerns about these risks, regulatory authorities require cell therapy products utilizing viral transduction to undergo monitoring and reporting of various product specifications, including number of vector integrations per cell and transduction efficiency.[10,11]
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