Transcriptomic Analysis of the Innate Immune Response to in vitro Transfection of Plasmid DNA

The innate immune response to cytosolic DNA is intended to protect the host from viral infections, but it can also inhibit the delivery and expression of therapeutic transgenes in gene and cell therapies. The goal of this work was to use mRNA-sequencing to reveal correlations between the transfection efficiencies of four cell types (PC-3, Jurkat, HEK-293T, and primary CD3+ T cells) and their innate immune responses to nonviral gene delivery. Overall, the highest transfection efficiency was observed in HEK-293T cells (87%), which upregulated only 142 genes with no known anti-viral functions. Lipofection upregulated a much larger number (n = 1,057) of cytokine-stimulated genes (CSGs) in PC-3 cells, which also exhibited a significantly lower transfection efficiency. However, the addition of serum during Lipofection and electroporation significantly increased transfection efficiencies and decreased the number of upregulated genes in PC-3 cells. Finally, while Lipofection of Jurkat and Primary T cells only upregulated a few genes, several anti-viral CSGs that were absent in HEK and upregulated in PC-3 cells were observed to be constitutively expressed in T cells, which may explain their relatively low Lipofection efficiencies (8-21%). Indeed, overexpression of one such CSG (IFI16) significantly decreased transfection efficiency in HEK cells to 33%.

Nonviral gene delivery vectors for transfection of the CAR gene for CAR-T cell therapy

The introduction of the CAR gene via retrovirus is the most widely used and most successful technique for CAR-T cell therapy. However, the limitations and risks associated with viral gene therapy continue to motivate the development of nonviral gene delivery technologies that are potentially safer, less expensive, and quicker. In terms of safety and efficacy, electroporation has emerged as the most promising non-viral technology, albeit more optimization testing and clinical trials are required before it can be directly compared to retroviral CAR gene delivery techniques. But these findings suggest that non-viral gene delivery to T cells (and other related cells, such as natural killer cells) is feasible and is quickly emerging as a viable alternative to CAR-T cell therapy and other gene therapies.

Nonviral Vehicles for Gene Delivery

Nonviral gene delivery (NVGD) is an appealing alternative to viral gene delivery for clinical applications due to its lower cost and increased safety. A variety of promising nonviral vectors are under development, including cationic polymers, lipids, lipid-polymer hybrids (LPHs) and inorganic nanoparticles. However, some NVGD strategies have disadvantages that have limited their adoption, including high toxicity and low efficiency. This review focuses on the most common NVGD vehicles with an emphasis on recent developments in the field.

A novel gene therapy for neurodegenerative Lafora disease via EPM2A-loaded DLinDMA lipoplexes

Aim: To develop novel cationic liposomes as a nonviral gene delivery vector for the treatment of rare diseases, such as Lafora disease – a neurodegenerative epilepsy. Materials & methods: DLinDMA and DOTAP liposomes were formulated and characterized for the delivery of gene encoding laforin and expression of functional protein in HEK293 and neuroblastoma cells. Results: Liposomes with cationic lipids DLinDMA and DOTAP showed good physicochemical characteristics. Nanosized DLinDMA liposomes demonstrated desired transfection efficiency, negligible hemolysis and minimal cytotoxicity. Western blotting confirmed successful expression and glucan phosphatase assay demonstrated the biological activity of laforin. Conclusion: Our study is a novel preclinical effort in formulating cationic lipoplexes containing plasmid DNA for the therapy of rare genetic diseases such as Lafora disease.