The linear covalently closed (LCC) DNA minivector (DNA ministring) is a non-viral gene delivery vector offering high transfection efficiency and is relevant to any DNA delivery application. The production system is simple, rapid, and scalable in vivo. The following protocol provides visual step-by-step instructions for the production of DNA ministrings.
Vi konstruerade linjära kovalent slutna (LCC) DNA minivectors som en icke-viral gen-leverans vektor alternativ som produceras via en enkel plattform in vivo. DNA ministrings har en ökad säkerhetsprofil och även effektivt leverera DNA last till målceller. Konventionella DNA-vektorer bär oönskade prokaryota sekvenser, inklusive antibiotikaresistensgener, CpG-motiv, och bakteriella replikations, vilket kan leda till stimulering av värdimmunologiska reaktioner. Biotillgängligheten av konventionella DNA-vektorer är också äventyras på grund av deras större molekylstorlek. Deras cirkulär natur kan också ge kromosomal integration, vilket leder till insertionsmutationer.
Bakteriella sekvenser skärs ut från DNA minivectors, vilket innebär att endast den intressanta genen (GOI) och nödvändiga eukaryota uttryckselement. Våra LCC DNA minivectors eller DNA ministrings, saknar immunogena bakteriella sekvenser; därför förbättra thEIR biotillgänglighet och GOI uttryck. I händelse av vektorn integreras i kromosomen, kommer LCC DNA ministring letalt störa värdkromosomen, varigenom avlägsnande av potentiellt farligt mutanten från prolifererande cellpopulationen. Följaktligen DNA ministrings erbjuder fördelarna med "minicircle" DNA samtidigt som man eliminerar risken för oönskade händelser vektor integration. I jämförelse med konventionella plasmider och deras isogena cirkulära kovalent slutna (CCC) motsvarigheter, DNA ministrings ingen signifikant bättre biotillgänglighet, transfektionseffektiviteten, och cytoplasmatiska kinetik – de kräver sålunda mindre mängder av katjoniska ytaktiva medel för effektiv transfektion av målceller.
Vi har konstruerat en en-stegs inducerbar in vivo-system för produktion av DNA-ministrings i Escherichia coli som är enkel att använda, snabb, och skalbar.
The goal is to produce scalable quantities of linear covalently closed (LCC) DNA minivectors using a simple and high efficiency one-step heat-inducible in vivo DNA ministring production system. DNA ministrings provide a safe and effective non-viral strategy to deliver DNA. They combine the safety of LCC vectors with the efficiency of DNA minicircles, and also offer a safer alternative to virus-derived vectors without compromising transfection efficiency.
In order for a transgene delivery system to be successful, the DNA vector must enter the target host cell and express the encoded transgene(s). There are several cellular barriers that need to be overcome in practice, particularly in mammalian systems. In order to avoid degradation by serum nucleases and immune detection by reticulo-endothelial system, the DNA vector must be bio- and immune-compatible with target system. Unprotected DNA is quickly digested by plasma nucleases and the plasmid membrane is composed of dense lipoprotein barriers so the DNA vector must be capable of rapidly crossing the plasma membrane of target cells. Once in the cell, vectors must traverse the cytoplasm and pass through the nuclear membrane to enter the nucleus for transgene expression. Non-viral gene delivery techniques focus on enhancement of tissue- and cell-targeting. However, the use of conventional plasmids with these techniques reduces transfection efficiency due to the presence of immunogenic bacterial sequences, which rapidly silences gene expression1,2. Conventional plasmids typically carry antibiotic resistance genes for maintenance in prokaryotic systems. However, these may produce potential adverse effects in human hosts or impart resistance to naturally occurring host flora via horizontal gene transfer effects. Conventional plasmids also contain dinucleotide CpG motifs2, which can trigger an unwanted immunostimulatory response, potentially reducing or silencing transgene expression.
As they are solely comprised of the eukaryotic expression cassette, DNA minivectors, such as DNA minicircles3, are a better alternative for gene delivery as they exhibit improved extracellular and intracellular bioavailability and improved gene expression due to their reduced size and absence of immunostimulatory prokaryotic elements4. The reduced vector size fares better with respect to resistance to shear forces associated with in vivo administration to a target site5. The higher copy number of the vector per unit mass requires less transfection reagent, thus decreasing toxicity. However, in the event of random vector integration into a host chromosome, circularly covalently closed (CCC) vectors, including both DNA minicircles and conventional plasmids, impart molecular continuity and therefore may lead to insertional mutagenesis, which can have devastating consequences6. Comparatively, integration of a linear DNA vector disrupts the chromosome and initiates cell death pathways, thereby removing the mutant cell from the proliferating cell population and preventing insertional mutagenesis7. Minimalistic immunologically defined gene expression (MIDGE) vectors8 and micro-linear vectors9 (MiLV) are LCC DNA vectors developed in vitro. MIDGE vectors have exhibited up to a 17-fold improved transgene expression in vivo compared to conventional plasmid DNA vectors11, and have demonstrated promising results in vaccine10 and cancer8 gene therapy. In addition, due to the torsion-free structure of the LCC minivector, less transfection reagent is required in comparison to the CCC supercoiled counterpart, thus reducing toxicity7.
Our enhanced LCC DNA vectors, DNA ministrings, have demonstrated superior expression efficiency and bioavailability7. Furthermore, DNA ministrings are produced on a one-step heat-inducible in vivo production system, an expedient and cost-effective alternative to MIDGE and MiLV LCC vectors, which require multiple steps in vitro. The DNA ministring production system to be demonstrated is a simple heat-inducible process performed in vivo, making it both cost-effective and easily scalable. This system exploits the Yersinia enterocolitica bacteriophage PY54-derived Tel/pal protelomerase recombination system12 to separate the minimal eukaryotic expression cassette from the prokaryotic plasmid backbone. We have engineered Escherichia coli cells (W3NN) to express Tel protelomerase under the control of the heat-inducible bacteriophage λ promoter, cI[Ts]85713. Upon expression, Tel protelomerase acts on pal target sites present within "Super Sequence" (SS) sites located on the precursor plasmid to yield LCC products, from which the DNA ministring can then be purified (Figure 1). The SS sites on the DNA ministring precursor plasmid also encode target sites for other recombinases including Cre recombinase (lox), TelN protelomerase (telRL) and Flp recombinase (FRT), thereby facilitating the production of isogenic LCC and CCC minivectors from one precursor plasmid. In addition, each SS site is flanked on both sides by SV40 enhancer (SV40e) sequences, which serve to improve nuclear translocation14. We have demonstrated elsewhere that successive addition of SV40e progressively confers corresponding increases in transfection efficiency7. The precursor plasmid (pDNA MiniString or pDMS) contains a polylinker (Figure 1) to facilitate insertion of any desired gene of interest in transcriptional fusion with the green fluorescent reporter (GFP).
The DNA minivector technology may be used in place of conventional methods for gene transfer, expression of reporter genes, and assessments towards the efficiency of transgene expression in eukaryotic systems. DNA ministrings combine the biocompatibility and increased transfection efficiency benefits of "mini" vectors with the superior safety profile of linear DNA vectors. Our robust one-step production platform rapidly and easily produces DNA ministrings for any gene transfer application and offers a higher safety profile.
We describe here a simple system for the production of LCC DNA ministrings using a one-step heat-inducible protocol, which requires no special equipment aside from that which is used in typical bacterial growth and manipulation. DNA ministrings are torsion-free, stable linear DNA expression cassettes, devoid of prokaryotic genetic elements. They may be used in place of conventional methods for gene transfer, expression of reporter genes, as well as in assessments towards the efficiency of transgene expression in eukaryotic systems. DNA ministrings combine the biocompatibility and increased transfection efficiency benefits of "mini" vectors with the superior safety profile of linear DNA vectors. Our robust one-step in vivo production platform rapidly and easily produces DNA ministrings for any gene transfer application without the need for multiple costly in vitro processes.
There are several critical steps to ensure optimal ministring production (Figures 2 and 3). Heat induction is the most critical step to ministring production. Complete lack of ministring production is most likely due to lack of heat induction (cells retained at 30 °C), where repression of protelomerase expression prevents conversion of precursor plasmid to LCC ministring. Following the heat induction protocol, expect a production efficiency of approximately 75%. Heat induction is optimal while cells are in log phase. Though there is no statistically significant difference in ministring PE when using cells in stationary phase ("Overgrown", Figure 3), we did observe almost a 50% decrease in overall plasmid yield. Yields will depend on the plasmid extraction protocol used. For optimal ministring production, trigger heat induction while cells are in log phase. Poor ministring production will most likely be a result of prolonged temperature upshift or insufficient temperature downshift. Prolonged temperature upshift is discouraged as this activates the E. coli heat shock response15, which may inhibit protelomerase activity and interfere with plasmid stability. Therefore, timing the temperature downshift is another critical step for efficient ministring production. Without additional incubation time at 30 °C, ministring production efficiency was found to be very poor ("Early Removal", Figure 3). This may be attributed to the amount of time required for Tel expression and activity. The originating prophage PY54 encoding Tel protelomerase normally infects Yersinia, which optimally grows around 28 °C12, indicating that 30 °C may also be more optimal for Tel activity.
The DNA minivector production system utilizes an in vivo platform to generate high quality bacterial sequence-free DNA minivectors. Modifications to the protocol may include altering the growth media to optimize plasmid and protein production in order to promote cell growth during the growth phase (TB instead of LB), or increase ministring production and conversion efficiency. For larger culture volumes (200 ml and greater), temperature downshift and incubation at 30 °C may be extended overnight without severe negative impact on ministring PE. We have included modifications in our protocol for 500 ml cultures as an example. Purification of DNA ministrings can be expedited through in vitro endonuclease digestion of the prokaryotic backbone. There are a number of endonuclease target sites (e.g., PI-SceI, PvuI, ScaI) available on the prokaryotic backbone of the precursor plasmid, which can be cut to expose open linear ends, allowing for in vitro degradation of the prokaryotic backbone. Isolation of ministrings from other vector species can also be achieved through anion exchange membrane chromatography16.
One current limitation associated with the LCC DNA vector production system is the need for purification of the DNA ministring from other LCC and CCC products. Although easily purified using standard plasmid isolation methods, the isolation step can still be a disadvantage in a large-scale setting. Separation of the ministring can be time-consuming using AGE if the ministring and the LCC prokaryotic backbone are too similar in size. Alternatively, anion exchange membrane chromatography may provide better separation of ministrings from CCC vector species16. Temperature upshift can be another potential limitation as high temperatures also activate the heat shock response in E. coli, which negatively affects recombinant protein yields17 and consequently reduce rates of plasmid to ministring conversion. We report elsewhere optimizations of the production platform to circumvent and/or mitigate such effects of heat shock18. We are also currently optimizing methods to eliminate or reduce LCC prokaryotic backbone contamination in vivo.
Chromosomal integration of the LCC DNA ministring disrupts the host chromosome and subjects the cell to apoptosis. Not only does this reduce potential negative consequences of insertional mutagenesis such as oncogenesis and silencing of adjacent genes, thereby improving its safety; the lethal effect can also serve as an ideal method for knock-in and gene replacement studies without the associated side effects and damage of integration. DNA ministring vectors can be applied towards the transfer and expression of genes for therapeutic proteins, RNA, antibodies, or antigens into a given target in vivo or replace a mal- or non- functional allele. The simple one-step heat-inducible in vivo production system allows DNA ministring vectors to be easily produced for clinical or industrial applications.
The authors have nothing to disclose.
The authors thank the Natural Sciences and Engineering Research Council of Canada (NSERC) for funding this work.
E. coli W3NN | Mediphage | strain for ministring processing: Nafissi & Slavcev, 2012 | |
pDMS.IRES.GFP | Mediphage | parent plasmid for ministring processing: also referred to as pDNA Ministring (pDMS) | |
BD™ Difco™ Dehydrated Culture Media: LB Broth, Miller (Luria-Bertani) | Fisher Scientific | 244620 | |
Fisher BioReagents™ Terrific Broth | Fisher Scientific | BP2468500 | |
BD™ Bacto™ Dehydrated Agar | Fisher Scientific | 214010 | |
Ampicilin | MP Biomedicals LCC | 2190147 | |
Ultrapure Milipore Water | Fisher Scientific | Z00Q0V0US | Model: Mili-Q Advantage A10 Water Purification System |
Surgical Gloves | VWR | (S) 82026-424, (M)82026-426, (L) 82026-428, (XL) 82026-430 | |
Fisherbrand™ Petri Dishes with Clear Lid Raised Ridge (100 x 15 mm) | Fisher Scientific | FB0875712 | |
30 mL KIMAX® Test Tubes | VWR | 89001-448 | |
Spectrophotometer Cuvette | Sarstedt | 67.74 | |
Sterile 15 mL Falcon Tubes | BD Falcon | 62406-200, | |
Sterile 50 mL Centrifuge Tubes | FroggaBio | 2930-S0 | |
Falcon™ Disposable Polystyrene Serological Pipets 10 mL | BD Falcon | 13-675-20 | |
Falcon™ Disposable Polystyrene Serological Pipets 25 mL | BD Falcon | 13-668-2 | |
Micropipettor (100-1000 μL) | FroggaBio | C1000-1 | |
Micropipettor (20-200 μL) | FroggaBio | C200-1 | |
Sterile Pipette Tips (100-1250 μL) | VWR | 89079-472 | |
Sterile Pipette Tips (1-200 μL) | VWR | 89079-460 | |
Fisher Scientific Economy Burner | Fisher Scientific | 03-917Q | |
Incubator Shaker | New Brunswick Scientific | M1352-00000 | |
Sterile Erlenmeyer Flask (125 mL) | Pyrex (Sigma Aldrich) | CLS4980125 Aldrich | |
Sterile Erlenmeyer Flask (250 mL) | Pyrex (Sigma Aldrich) | CLS4980250 Aldrich | |
Sterile Erlenmeyer Flask (500 mL) | Pyrex (Sigma Aldrich) | CLS4980500 Aldrich | |
Spectrophotometer | Thermo Fisher Scientific | 335901 | Model: Genesys 10 Vis |
Floor Centrifuge | Beckman Coulter | 369001 | Model: Avanti J-E |
Benchtop Centrifuge | Beckman | 362114 | Model: GS-6R |
Fisher Scientific Analog Vortex Mixer | Fisher Scientific | 02-215-365 | |
1 kb DNA Ladder | New England BioLabs | N3232S |