MiniVec™ Plasmid
VectorBuilder’s proprietary MiniVec™ plasmid offers a miniaturized backbone, providing remarkable efficacy, safety, and manufacturability for applications in genetic medicine, vaccine formulations, and food technology. Compared to traditional plasmids, MiniVec™ plasmids have higher plasmid manufacturing yields and transgene expression, enhance performance in various applications such as virus packaging, CRISPR gene editing, and transposon delivery, and demonstrate a better safety profile. The MiniVec™ backbone supports antibiotic-free and supplement-free selection and can be applied across diverse expression systems, including lentivirus, AAV, in vitro transcription (IVT), non-viral regular plasmids, and transposon systems.
Explore how MiniVec™ can streamline your gene delivery solutions.
Talk to our experts today!MiniVec™ Technology
The MiniVec™ plasmid operates within the exclusive MiniHost™ bacterial host strain, which is an engineered E. coli strain containing both a growth inhibitor gene downstream of a SeqA sequence as well as an antidote gene encoding a protein that can neutralize the growth inhibitor to support cell proliferation. Expression of the antidote gene is inducible by a specific media supplement.
Upon removal of the supplement and transformation of a MiniVec™ plasmid into MiniHost™ cells, a SeqB gene carried on MiniVec™ produces a small noncoding RNA complementary to the SeqA region of the growth inhibitor transcript, suppressing translation of the growth inhibitor and therefore allowing cell proliferation. As a result, only the successfully transformed host cells proliferate robustly to produce high copies of the MiniVec™ plasmids. This technology completely eliminates the need for antibiotics and supplements, which not only greatly simplifies large-scale fermentation and manufacturing scalability, but also significantly enhances safety of the final drug or food product.
Figure 1. Mechanism of selection with MiniVec™ backbone.
Substantial Increase in Plasmid Yields
In both lab- and industrial-scale fermentation conditions, MiniVec™ exhibits significantly improved plasmid production and manufacturing yields compared to traditional plasmids in all tested gene delivery systems. This marked increase is due to several key mechanisms including a high-copy-number favored fermentation, more efficient replication, and a reduced metabolic burden.
Figure 2. MiniVec™ exhibits increased plasmid yields compared to traditional plasmids. Measurement of plasmid yields from (A) lab-scale (200 ml) or (B) industrial-scale (2.7 L) fermentation of E. coli cultures under the same conditions transformed with traditional or MiniVec™ plasmids from different expression systems.
Enhanced Efficiency Across Various Vector Systems
In vitro validation
As a result of its minimal bacterial backbone, MiniVec™ reduces the risk of an immune response in mammalian cells, which minimizes host cellular stress and allows for increased cellular uptake and trafficking. MiniVec™ has been compared to traditional backbone in a broad range of gene delivery applications and consistently demonstrates significant improvements in efficiency and efficacy.
- Transient transfection
- Virus packaging
- Transposon delivery
- CRISPR genome editing
Figure 3. Comparison of transient gene expression using traditional and MiniVec™ plasmids. HEK293T cells were transfected with equal molar MiniVec™ or traditional plasmids encoding EGFP. Measurement of EGFP expression via flow cytometry and representative fluorescence microscopy images were taken 48 hours post-transfection. An mCherry expressing plasmid was co-transfected as the transfection control. Average mean fluorescence intensity (MFI) of all viable cells was calculated.
Figure 4. Comparison of lentivirus packaging using traditional and MiniVec™ plasmids. (A) Higher functional titer (qPCR-based) was achieved using MiniVec™ packaging plasmids than traditional plasmids with same packaging scale. (B) Comparison of EGFP expression in HEK293T cells through equal volume transduction of lentivirus in-parallel produced using traditional vs. MiniVec™ plasmids. Average mean fluorescence intensity (MFI) of all viable cells was calculated.
Figure 5. Comparison of transposition efficiency of traditional and MiniVec™ plasmids. HEK293T cells were transfected with traditional or MiniVec™ plasmids encoding EGFP utilizing the (A) piggyBac or (B) Sleeping Beauty system in equal molar conditions. Representative fluorescence images were taken and mean fluorescence intensity measurements were recorded by flow cytometry at early passage and late passage when the corresponding control sample (not shown) had negligible fluorescence left.
Figure 6. Comparison of CRISPR-mediated EGFP knockin using traditional and MiniVec™ plasmids. HEK293T cells were transfected with either traditional- or MiniVec™-based hCas9 and donor plasmids expressing EGFP under equal molar conditions to examine the efficiency of EGFP genome integration at the AAVS1 locus through homology-independent targeted insertion (HITI). Mean fluorescence intensity of cells was measured by flow cytometry at indicated time points until negligible fluorescence was left in the control sample (not shown).
In vivo validation
This improved efficiency also translated to in vivo applications, where administration of MiniVec™ plasmids resulted in increased and prolonged transgene expression in gene delivery models and successfully stimulated acute and long-term immune responses in a vaccine delivery model. Importantly, all in vivo studies indicated no physical or physiological adverse effects, further supporting utilization of MiniVec™ in gene therapy and vaccine applications.
- Gene delivery
- Vaccine delivery
Figure 7. Comparison of transgene expression of traditional and MiniVec™ plasmids in vivo. Equimolar plasmids (CAG>Luc2) were administered in mice via (A) intravenous or (B) intramuscular injection. Luciferase expression was measured at the indicated time points.
Figure 8. Comparison of immune response to naked DNA vaccination between traditional and MiniVec™ plasmids expressing the COVID-19 spike protein. BALB/c mice were injected intramuscularly with equimolar doses of either traditional- or MiniVec™-based vaccines, a negative control (PBS), or vehicle control (empty traditional or MiniVec™ plasmids) 3 times at 2-week intervals. (A) Specific antibody titer was measured in blood drawn from the animals 2 weeks after each injection, before the next immunization. On day 42, splenocytes were harvested and culture supernatant of the splenocytes was used to measure (B) total IFN-γ secretion upon spike protein stimulation.
Distinctly Improved and Reliable Safety Profile
In addition to its demonstrated benefits in diverse applications across the cell and gene therapy landscape, VectorBuilder’s innovative MiniVec™ platform offers an evidently safer alternative to traditional antibiotic-dependent plasmids in the GMP production of drug products by removing the potential of antibiotic residues, mitigating the risk of horizontal gene transfer, and ensuring the purity of the final product. The excellent safety profile is also confirmed in separate in vivo validations, where toxicology analyses of animals administered with MiniVec™ plasmids revealed no adverse effects on animal health and empty MiniVec™ constructs elicited no immune response. Importantly, design principles of the system are in compliance with regulatory standards put in place by the FDA and EMA for the manufacturing of therapeutics.
How to Order
Explore how MiniVec™ can streamline your gene delivery solutions.
Talk to our experts today!Resources
Further Reads
CRISPR This Way: Choosing Between Delivery Systems
Keywords: CRISPR delivery, plasmid, virus, mRNA, RNP
A comparison of delivery systems to help you choose the optimal method for your CRISPR experiments, from research applications to clinical use.
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