PlasmidFactory’s small but powerful minicircle DNA:
From parental plasmid to highly efficient minicircles, we focus on what really matters
Introducing an advanced, powerful minicircle DNA technology – PlasmidFactory‘s bespoke minicircle DNA is tailored to customer requirements, and produced using self-developed and patented minicircle technology.
A plasmid containing the Gene of Interest – GOI, provided by the customer, serves as starting material. This is then inserted into the parental plasmid, and subsequent intramolecular recombination leads to the formation of the minicircle DNA molecule, which consists almost exclusively of the GOI. The smaller size of minicircles compared with standard plasmid vectors makes for greater transfection and expression efficiency, while a decrease in the number of CpG motifs reduces the risk of immunogenicity. Other key advantages compared to plasmids include more stable transgene expression and reduced DNA toxicity. Our minicircle DNA is available in large scale and HQ grade.
A key inventive step for us was to use Minicircle DNA to encode the CAR which reduces the amount of DNA from a conventional plasmid which is 8-9 kbp to just 4 kbp …
Minicircle features
- No bacterial backbone
(e.g. reduced CpG motifs, no replication origin) - Free of any bacterial selection markers
- Free of antibiotic resistance genes
- Free of antibiotic residues
- Smaller size compared to plasmid DNA
- Monomeric supercoil
- Almost no cargo size restrictions
- Available on a large scale
Minicircle advantages
- High expression efficiency
- Reduced transgene silencing
- Increased yield – Reduced costs
- No retro packaging in AAV
- Reduced DNA toxicity
- Lower safety risk: already meets future regulatory requirements
- Successful in clinical CAR-T cell research
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Insights
Minicircle vs. Plasmid
Minicircles have by now been tested in approximately 20 efficiency studies. In all cases, gene expression levels from the minicircle equaled or, more frequently, significantly exceeded those in the corresponding plasmid with the same expression cassette.
Expression has been tested in various cell types, tissues, and animals, using a range of transfection methods (e.g. electroporation, sonoporation, lipofection, magnetofection, hydrodynamic injection, jet injection). The McBox® by PlasmidFactory comprises the minicircle and the corresponding conventional plasmid and is very well suited to comparative laboratory experiments.
It is important to note that only equimolar comparison (same number of DNA molecules – minicircles or plasmids) is advisable.
PlasmidFactory’s minicircle DNA is tailored to customer requirements.
Minicircles (for AAV production or with reporter genes) are available at short-term as In-Stock products in Research grade quality.
Comparison of our quality grades: Minicircle
We provide different quality levels for research and (pre-) clinical studies to ensure optimal adaption to your application. Our High Quality grade* minicircles are particularly suitable for GMP-compliant production of viral vectors and RNA, for example.
*HQ: High Quality Grade is produced in accordance with EMA guideline CHMP/BWP/2458/03 as the highest non-GMP quality standards **On request| Grades table as PDF | Research grade-MC | High Quality grade*-MC | GMP grade-MC |
|---|---|---|---|
| Characteristics | Applications | Applications | Applications |
| Guaranteed amount of Minicircle DNA | ** | ||
| Adjustment of DNA concentration included | |||
| Customized filling included | ** | ||
| Certified quality incl. QC Report | |||
| Storage of glycerol stock of parental plasmid and retain samples for repeat orders | |||
| Antibiotics-free fermentation | |||
| Cultivation media according to EMA/410/01 rev.3, 2011/C 73/01 (TSE/BSE certificate) | |||
| All raw materials according to EMA/410/01 rev.3, 2011/C 73/01 (TSE/BSE certificate) | |||
| Characterized and documented cell bank and pilot cultivation | |||
| Verified removal of bacterial endotoxins (LPS assay) | |||
| Removal of RNA and proteins | |||
| Specific removal of bacterial chromosomal DNA and oc-forms | |||
| CGE analysis (ccc-supercoiled vs. oc plasmid topologies) | |||
| Extended specification | ** | ||
| Documentation acc. to GMP/GMP principles | |||
| Dedicated lab | |||
| QM system applied | |||
| Spatially separated upstream and downstream processes | |||
| Single use equipment | |||
| Compliant with applicable GMP-guidelines and GMP certified |
EMA Guidelines
The term “High Quality” is consistent with the meaning embodied in EM(E)A document CHMP/BWP/2458/03 (“Guideline on Development and Manufacturing of Lentiviral Vectors”); EMA document EMA/CAT/80183/2014 (“Guideline on the quality, non-clinical and clinical aspects of gene therapy medicinal products”); and EMA document EMA/246400/2021 (“Questions and answers on the principles of GMP for the manufacturing of starting materials of biological origin used to transfer genetic material for the manufacturing of ATMPs”). PlasmidFactory seeks to ensure maximum safety and the highest quality DNA, although the manufacturing itself is not aligned to GMP standards.
… The gene transfer rate that we accomplish in primary human T cells is quite impressive and is always in excess of 50-60 %, which is 5 to 6-fold higher than with plasmid DNA.
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I would like personalised advice on the following topic …
- Manufacturing of customized plasmid DNA
- Manufacturing of customized minicircle DNA
- In-Stock products for AAV production
- In-Stock products (reporter-plasmids/-minicircle)
- cooperation opportunities
I would like a specific quote for the following product
- Customized DNA
- AAV plasmids und minicircles
- reporter gene plasmids and minicircles
- pEPito plasmids
- Molecular Size Markers
- CGE Service
- Storage Service
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