A new publication by scientists at the Cell and Gene Therapy Catapult in the UK cites the use of HemaCare-sourced CD3+ T cells for their cancer immunotherapy research.
The successful commercialization of gene-modified cell therapies relies in part on being able to manufacture a consistent product. Many cell and gene therapy developers use lentiviral vector delivery systems to impart the therapeutic gene modifications necessary to making their therapies effective.
Lentiviral vectors are used to deliver genetically modified versions of genes that scientists hope will imbue cells with therapeutic capabilities. However, the rate at which cells successfully incorporate lentiviral vectors is difficult to control. Inefficient or variable vector incorporation impacts the efficacy of the downstream therapeutic product, which increases the risk of batch failure during manufacturing, and potentially poses a risk to patients.
The goal of this study was to develop a rapid, sensitive method of validating vector incorporation, to assure that downstream product quality is consistent. To accomplish this, the scientists first needed to prove they could discriminate between transduced cells which have incorporated copies of the vector and non-transduced cells, or cells with a low vector copy number (VCN). They would then need to validate that VCN data from their single-cell technique was consistent with conventional analysis methods which screen total cell populations.
The scientists decided to test their methodology using a T cell immunotherapy model transduced with lentiviral vector coding for green fluorescent protein (GFP), so that they could easily track assay results.
CD3+ T cells used as starting material for the project were isolated from HemaCare healthy donor leukopaks, and validated as having >95% viability and >95% purity. Following purification, the researchers also established a cryopreserved cell bank of CD3+ cells for future use. Cell and Gene Therapy Catapult scientists have relied on HemaCare for years as a starting material supplier. Donor restrictions in the UK make it difficult to obtain locally sourced human donor material, and HemaCare is well known for maintaining one of the world’s largest recallable donor networks.
In this study, CD3+ T cells were transduced with lentiviral vector and expanded in culture. Single cells isolated by serial dilution were loaded onto a chip via microfluidic technology. Genetic material from each single cell preparation was pre-amplified to increase the genetic signal strength, then analyzed by a technique known as “digital droplet PCR”. This technique enables the reporting of the absolute copy numbers of a genetic mutation per mL of fluid.
By using a complex statistical analysis method developed in house, the researchers were able to demonstrate that their single cell analysis method closely replicated total cell population transduction efficiency data collected through conventional live imaging and FACs analysis techniques. The new single cell approach provides a more granular analysis of transduction variability across a given cell population and does not need to rely on specific antibody availability, but rather on primers specific to the therapeutic gene being evaluated.
Cellular therapies are extremely complex and expensive to develop. The intrinsic variability of living cells complicates manufacturing and development, creating a need for equally complex analytical tools and quality assurance oversight to ensure that cell-based therapeutics are consistent in their composition and efficacy. The ability to analyze cell-to-cell variability will support optimized manufacturing methods and more cost-efficient regulatory compliance.
Santeramo I., et al. Vector Copy Distribution at a Single-Cell Level Enhances Analytical Characterization of Gene-Modified Cell Therapies. Molecular Therapy: Methods & Clinical Development. (17); 944-955. June 2020.