FAQs About MiniVectors

What are MiniVectors?

MiniVectors are small DNA circles that can be as small as ~336 bp and as large as clients need (there is no upper limit of DNA length). MiniVectors offer advantages in many applications, most notably as substrates for biochemical and biophysical studies of DNA, and the proteins and drugs that act on DNA, and they have proven to be superior non-viral gene delivery vectors.

What makes MiniVectors different from non-viral vectors

There are several important qualities of MiniVectors. First is MiniVector purity. Our patented purification process completely removes parent vector and unwanted sequences. Clients have reported problems with these contaminants in minicircle preps from other sources. Second is the small size of MiniVectors, as small as 336 bp (exactly 32 turns of the DNA double helix). No other vector can be made that small. Third is the degree of DNA negative supercoiling. Through a patented process, MiniVectors are more supercoiled (making them more compact and thus more likely to transfect cells) than other vectors.

How do MiniVectors compare to other nucleic acid delivery methods?

Like tools in a toolbox, each vector has a specific use and the client must assess the specific need before choosing the most appropriate vector. For clients desiring a straight path to the clinic, MiniVectors are an easy choice. For some applications, a one-time delivery using a viral vector may be best. siRNA, especially for screening and experimental purposes, is useful for initial experiments, but clinical applications with siRNA may prove prohibitively expensive. MiniVectors are most similar to plasmids. Both are cheap and easy to ship and store. Plasmids, however, contain unwanted sequences, cannot survive aerosolization or human serum, and can be toxic. Furthermore, transfection efficiency of plasmids is poor for many clinically relevant cell-types. Plasmids MiniVectors survive aerosolization and human serum, are not toxic, and transfect clinically relevant cells and animal models.

How small can MiniVectors be?

As we have published (Fogg et al. 2006), through a special patented process we can make circles as small as 336 bp (32 exact turns of the double helix) with very high yield.

Why is it important for MiniVectors to be small?

For biophysical and biochemical studies of DNA, proteins, or drugs that act on DNA, 336 bp is ideal. Approximately the size of a topoisomerase, MiniVectors allow quantitative biophysical and biochemical experimentation. Furthermore, large quantities of specific supercoiling levels (topoisomers) can be generated, allowing precise supercoiling dependence of various proteins or drug binding to be assessed.

For transfecting primary cells, stem cells, live animals, and other cell types that are difficult to transfect, small supercoiled MiniVectors are the best option; larger vectors either fail or cause significant DNA vector length-dependent toxicity.

How big can MiniVectors be?

There is no upper limit for how long a DNA sequence a MiniVector can be. Whereas the specific size-related advantages are lost when MiniVectors are large, the advantages afforded by a vector with just the desired sequence remain.

What types of cells can be transfected with MiniVectors?

So far every cell type tested, including in live animals, are transfected with MiniVectors. Published validated cell types include adhesion cells 293FT (transformed human embryonic kidney cell line), suspension cells Jurkat (human T-lymphoma/leukemia cell line), suspension cells Karpas 299 (human anaplastic large cell lymphoma cell line). Unpublished validated cell types include adhesion HeLa cells (human cervix adenocarcinoma cell line), adhesion A549 cells (human adenocarcinoma alveolar basal epithelial cell line), adhesion U2OS cells (human bone osteosarcoma cell line), human and mouse hematopoietic stem cells, macaque T cells, retinas and lungs of mice, and zebrafish embryos.

Why do MiniVectors transfect cells better than plasmids?

We still do not yet understand exactly why smaller vectors transfect better than larger ones. Logically, the smaller the size, the easier it should pass through cell membranes and the nucleus. Electron micrographs of plasmids and MiniVectors (shown below) provide additional hints. Plasmid DNA of typical lengths are highly hetero-disperse and heterogeneous. It is hard to imagine how this entangled net of DNA can penetrate cells. In contrast, MiniVectors are highly mono-disperse and homogeneous.

minivectors

Why do MiniVectors survive aerosolization?

MiniVectors are resistant to sheer forces used to make aerosol because their increased supercoiling and small size makes their “radius of gyration” (basically their effective diameter) smaller than the threshold size that gets shredded by the high pressures. No other vector survives this process. You can read more details about this important finding in Catanese et al. 2012.

Why do MiniVectors survive human serum?

athogen DNA. This natural protection mechanism makes it difficult to deliver nucleic acids through the blood. We were surprised to discover that a 386 bp MiniVector survived commercially available human serum that rapidly digested plasmid and synthetic siRNA (see Zhao et al. 2011). Whether there is a size threshold effect similar to that for surviving aerosolization or a vector length-dependent effect is not known. We hypothesize that either other serum proteins may bind and protect the smaller vectors or simply that longer vectors are more likely to encounter a nuclease than shorter ones.

Why are MiniVectors less toxic than other vector delivery methods?

Twister created the safest vector on the market. MiniVectors are not contaminated with the “parent” vector they were created from or the leftover sequences that contain all the toxic bacterial sequences. We take several steps to remove all of these problematic sequences. According to the regulatory agencies in most countries, it is no longer acceptable to introduce antibiotic resistance genes to patients during treatment. Our discarded leftovers contain all the bacterial sequences, including the genes encoding antibiotic resistance. There is additional DNA plasmid length-dependent toxicity that we do not yet understand.

How are MiniVectors manufactured?

Harmless gut bacteria grown in large batches have been engineered by a patented process to delete all the unwanted sequences from a plasmid. We then purify the remaining MiniVectors that contain only the desired DNA sequence.

What quality control procedures/criteria are applied during the manufacture of MiniVectors?

Twister requires the most rigorous quality control assessments and documents each important step, including purity; these quality control data are included in each shipment.

What is the best approach for use of MiniVectors for transfection?

Our clients have successfully transfected MiniVectors using many different transfection reagents or electroporation. We have achieved similar success with several commercially available products. Client choice of reagent or transfection method depends upon cell type or tissue type to be transfected.

Can Twister Biotech perform experiments for me?

Twister Biotech is glad to work with each client for their individual needs. We advise clients regarding the design of their MiniVector, but we do not often perform such experiments. Please call 713-876-0177 or send us a note and we will figure out how we can help you.

For additional questions contact us.