Enhancing Gene Transfer Efficiency in Viral and Non-Viral Vectors for Therapeutic Delivery

Background & Aim: Maximizing the efficacy of therapeutic delivery while minimizing risks is key to improving therapeutic development. Viral vectors, such as lentivirus and adeno-associated virus (AAV), are noted for their high efficiency and stable integration, but pose risks such as immunogenicity and insertional mutagenesis. Non-viral methods, including lipid-based nanoparticles (LNPs) and CRISPR/Cas9-based delivery offer safer alternatives with lower immunogenic risks; however, the typical payload can have limited bioavailability. Each of these methods has a critical component of either the delivery vehicle or payload which governs experimental success. Methodology: Here we describe optimized workflows to minimize screening time while maximizing successful research results across the top three delivery vectors of choice: lentivirus, AAV, and mRNA. Results: An efficient lentiviral packaging method with robust packaging produces high-titer lentiviral vectors, tailored for difficult-to-transduce cells and high-throughput packaging in 96-well plates suitable for CRISPR screening. Our results show that proprietary plasmid synthesis and preparation techniques maintain ITR integrity through AAV packaging and improve gene delivery. Finally, a streamlined gene-to-mRNA workflow to overcome limitations in mRNA production. This includes rapid synthesis with optimization of long polyA regions in mRNA for enhanced LNP encapsulation and efficient delivery, in vitro transcription, and novel sequencing of mRNA constructs for complete characterization of critical components such as the polyA tail. Conclusion: Lentiviral vectors are powerful cell engineering tools used to infect target cells followed by integration of the gene of interest into target cell genome. For difficult-to-infect target cells including hematopoietic stem cells and neuronal cells, high viral titer is critical for efficient transduction. The ITR regions in AAV plasmids are notoriously unstable, prone to full or partial deletions during propagation in bacteria, thus high-quality plasmid synthesis and preparation is required to prevent mutated ITRs, and mixed population plasmid DNA preparations. Lastly, sequence accuracy of the DNA template and integrity of transcribed mRNA are crucial factors in determining the success of downstream experiments for mRNA therapies, as defined by high quality synthesis and sequencing of the mRNA constructs.