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Enhancing Gene Delivery With Novel Miniaturized Plasmids: miniVec

Scientist on March 27, 2024
in Tech Snapshot

This blog post was written by VectorBuilder, a global leader in gene delivery technology.

Traditional plasmids, commonly used in molecular biology, have drawbacks that limit their use in cell and gene therapy. One major issue is the reliance on antibiotic resistance markers for selection during plasmid production, which poses concerns related to antibiotic overuse and the potential transfer of resistance genes to bacteria in the environment or in humans. To combat this problem, VectorBuilder’s team recently developed miniVecTM, a novel antibiotic-free and supplement-free miniaturized plasmid, to allow drug developers to enhance the safety, efficacy and manufacturability of their therapeutic plasmids for genetic medicines. Comparing to traditional plasmids, miniVec™ plasmids have higher plasmid manufacturing and virus packaging yields, improved transgene expression and an enhanced safety profile. miniVec™ plasmids can be applied across diverse vector systems, including lentivirus, AAV, in vitro transcription (IVT), non-viral regular plasmids, piggyBac and Sleeping Beauty.

Minimal backbone and antibiotic-free selection

Antibiotic-free selection offers multiple benefits, including enhanced intrinsic safety and the ability to manufacture therapeutic plasmids without antibiotics. VectorBuilder’s miniVec™ technology further distinguishes itself with its supplement-free cell survival mechanism, eliminating the need for additional supplements in culture media during fermentation.

Figure 1. Mechanism of antibiotic-free and supplement-free selection with miniVecTM.

Figure 1 depicts how the miniVec™ system works. As a key component of the system, miniHost™ is an engineered E. coli strain containing a growth inhibitor gene. After transforming with the miniVec™ plasmid, miniHost™ cells can be cultured in regular LB medium without the need for antibiotic-based selection. The VecSeqB transcribed from the miniVec™ backbone can block the growth inhibitor gene translation through RNA-RNA complementation with the VecSeqA that is positioned upstream of the growth inhibitor transcript. As a result, only the successfully transformed host cells proliferate robustly to produce high copies of the miniVec™ plasmids.

Improved plasmid yields

The miniVecTM plasmids offer several advantages beyond the antibiotic-free selection. Firstly, their high-copy-number favored fermentation, efficient replication and reduced metabolic burden result in increased plasmid yields across a variety of expression systems.

Figure 2. miniVec™ exhibits increased plasmid yields compared to traditional plasmids. Measurement of plasmid yields from lab-scale fermentation of E. coli cultures under the same conditions transformed with traditional or miniVecTM plasmids from different expression systems.

Enhanced transgene expression

In addition, miniVec™ plasmids have been validated for enhanced transgene expression in vitro compared to traditional plasmids. The minimal bacterial backbone, which mitigates the risk of an immune response in mammalian cells, can additionally minimize host cellular stress and exhibit increased cellular uptake and trafficking.

Figure 3. miniVec™ exhibits increased GOI (e.g., EGFP) expression compared to traditional plasmids in HEK-293T cells 48 hours after equal molar transfection as measured by flow cytometry and fluorescence microscopy. An mCherry expressing plasmid was co-transfected as the transfection control.

miniVec™ plasmids are further validated for enhanced transgene expression in vivo compared to traditional plasmids. Due to these features, miniVec™ is an ideal candidate for gene therapies and DNA vaccines.

Figure 4. miniVec™ exhibits increased and prolonged luciferase expression compared to traditional plasmids in mice. Plasmids (CAG driving Luc2) were administered (A) intravenously or (B) intramuscularly at equal molarity.

Efficient transposon delivery

miniVec™ is an ideal choice for transposon systems due to its streamlined design, reduced metabolic burden on host cells, and enhanced safety profile. These features allow miniVec™ to achieve increased transposition efficiency in cells.

Figure 5. Comparative analysis of transposition efficiencies between traditional and miniVec™ plasmids for (A) piggyBac and (B) Sleeping Beauty systems in HEK-293T cells as measured by flow cytometry.

Safer source materials for virus packaging

miniVec™ plasmids offer a safer alternative to traditional antibiotic-dependent plasmids in the GMP production of recombinant viruses, reducing the risk of potential antibiotic residues in the final product and mitigating the risk of horizontal gene transfer. This enhances the safety profile of the GMP process, aids in compliance with regulatory standards and ensures the purity of the final product. In addition, miniVec™ packaging plasmids have been shown to achieve higher titer for virus packaging than traditional plasmids.

Figure 6. Comparison of lentivirus packaging using traditional and miniVec™ plasmids. (A) Higher lentivirus titer is achieved using miniVec™ plasmids. Functional viral titers were measured by qPCR post-transduction. (B) Comparison of EGFP expression through equal volume transduction of lentivirus in-parallel produced using traditional vs. miniVec™ plasmids.

Partner with VectorBuilder for your miniVecTM project

In conclusion, the unique attributes of VectorBuilder’s miniVec™ plasmid position it as the optimal choice for cell and gene therapy developers. Its miniaturized backbone not only enhances efficacy and safety but also improves manufacturability across various systems. The remarkable increase in plasmid manufacturing and virus packaging yields, coupled with improved transgene expression and an enhanced safety profile, underscores miniVec™’s superiority over traditional plasmids. Furthermore, the versatility of the miniVec™ backbone, supporting antibiotic-free and supplement-free selection, enables its seamless integration into diverse expression systems, including lentivirus, AAV, IVT, non-viral regular plasmids, piggyBac and Sleeping Beauty. Overall, miniVec™ emerges as an innovative and comprehensive solution, meeting the evolving needs of cell and gene therapy developers. To get started visit: https://en.vectorbuilder.com

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