(337cf) Increased T-Cell Transfection By mRNA Lipid Nanoparticles for Cell Therapy Manufacturing

Chimeric Antigen Receptor (CAR) T-cell therapy has provided life-saving alternatives when conventional cancer treatments were not successful. However, the availability to patients remains limited due to complex and costly biomanufacturing. Genetic engineering of T-cells to express CAR is commonly achieved using viral vectors, that add to the cost and have safety concerns, such as insertional oncogenesis, immunogenicity, and cytotoxicity. Non-viral alternatives would be preferred but a major barrier is their far lower efficiency of gene delivery compared to viral methods. We developed a process that greatly improved transfection efficiency with lipid nanoparticles (LNPs), resulting in increased yields on LNPs of genetically engineered cells.

Methods

Human T-cells were transfected in vitro with green fluorescent protein (GFP) messenger RNA (mRNA) encapsulated in LNPs using a conventional protocol or the developed process. The percentage of GFP+ cells was determined using flow cytometry. Cell concentrations and viability were determined using CEDEX HiRes Analyzer with trypan blue viability dye. LNP size, polydispersity index and zeta potential were measured using dynamic light scattering. LNP encapsulation efficiency was determined using RiboGreen assay.

Results

Using conventional transfection, we identified an optimal mRNA-LNPs dose that resulted in up to 69% GFP+ cells and 55% GFP+ cells on average (n = 7 donors). Using a novel process developed in this study, we achieved up to 57% GFP+ cells and 49% GFP+ cells on average (n = 8 donors), while using 40-fold less mRNA per million cells. This corresponded, on average, to a 31-fold increase in the number of GFP+ cells per ng mRNA using the developed method compared to conventional protocol at the dose with the highest %GFP+ cells. Transfection with mRNA-LNPs also maintained high cell viability. At 24 h post-transfection, cell viability was 71% in top-performing conventional protocol and 76% using the developed process.

Implications

Transient (i.e., short-term) transfection with mRNA is being explored to generate CAR T-cells with reduced risk of adverse side effects and can be useful in other therapeutic applications, such as to treat cardiac injury. For applications that require long-term expression, such as currently approved CAR T-cell therapies, CRISPR/Cas9 RNA can be encapsulated in LNPs instead of mRNA. Overall, the developed process has the potential to address a barrier to more widespread implementation of LNPs use vs. viral vectors for a wide range of cell therapy applications. This could improve treatment safety and greatly reduce manufacturing costs, thus making these life-saving therapies more widely available to patients in need.

Research Interests

The presenting author’s research interests include bioprocess engineering to address the specialized needs of cell therapy manufacturing and immune cell engineering to treat diseases. The author has also published a review on the impacts of spaceflight on human health and is interested in utilizing microgravity platforms for life sciences and pharmaceutical experiments.