Designing Tailor-Made plasmid DNA
The conventional approach to plasmid DNA design and construction is not amenable to fine-tuning the structure and properties of these molecules. A regular restriction ligation is inserted into a pre-existing plasmid with a backbone that cannot be easily modified. Often an optimum molecule is not obtained, and the entire process must be repeated to see if insertion at a different location may yield a plasmid with better properties.
To overcome these significant limitations, we developed a unique and innovative plasmid DNA assembly technology e-Zyvec® that makes custom drawing of plasmids from scratch possible. The sequence for the gene of interest is combined with the various required functions of the plasmid, and all of these components are assembled together in one step. Our Draw your plasmid online software is based on the unique and innovative plasmid DNA assembly technology e-Zyvec® that makes custom drawing of plasmids intuitive and fast.
Discover what makes e-Zyvec® technology unique
The Draw your plasmid online software determines and optimises required genetic features and their predicted arrangement, i.e. their position and orientation in the desired plasmid. The specific features are matched to “DNA bricks” or templates of an extensive library within the software. For components that do not have matching templates—typically the client’s gene of interest, the software enables the design of new DNA bricks so they will be fully compatible with existing templates. The software also determines the optimum of several standardized assembly protocols for production of each specific plasmid DNA.
In this manner, the DNA bricks and the conditions for assembly are newly calculated for every project to ensure optimal plasmid DNA production. The controlled reaction using the selected DNA bricks and the chosen assembly method results in the production of plasmids that contain only the required genetic features (Fig. 1).
Synthesis and Screening
Synthesis of physical DNA bricks are generated using high-fidelity PCR on appropriate DNA templates prior to plasmid assembly. The optimized assembly conditions allow the routine assembly of up to 12 different DNA bricks, each from several hundred to 8000 bp, in a seamless fashion into circular plasmids ranging from 4 to 15kb.
Given that each single molecule is obtained from a unique mix of DNA bricks, several plasmids can be assembled in parallel by varying the mix of DNA bricks contained in each vial. Such a series of plasmids with different structures and properties can be rapidly screened to identify the optimum design. With this approach, the plasmid engineering service reduces time and cost of plasmid DNA design and construction, while ensuring state-of-the art and personalized plasmid DNA design for each specific customer application.
Optimized for Viral Vectors
Many diseases result from genetic aberrations that are inherited or result from exposure to carcinogenic substances or radiation. While some genetic disorders, such as Down Syndrome, are caused by chromosomal anomalies, many are due to mutations in one or more genes. In the latter case, the nucleotide sequence of the gene is altered such that the gene cannot properly fulfill its physiological function. For instance, a protein may be expressed at too high or too low levels, not at all, or in an inactive state.
Several approaches to gene therapy are being exploited today, both in research and in approved therapies. Some involve in vivo delivery of new genetic material, such as Luxturna® (voretigene neparvovecrzyl, Spark Therapeutics) for the treatment of vision loss due to a variety of inheritable retinal dystrophies and Zolgensma® (onasemnogene abeparvovec-xioi, Novartis) for the treatment of babies born with spinal muscular atrophy.
With chimeric antigen receptor (CAR) T-cell therapies, patient cells are genetically modified ex vivo and these modified cells are administered back to the patient. Examples include Tecartus™ (brexucabtagene autoleucel, Kite Pharma, Inc.), Kymriah™ (tisagenlecleucel, Novartis) and Yescarta™ (axicabtagene ciloleucel, Kite Pharma, Inc.). Several treatments leveraging gene editing technologies such as CRISPR-Cas9 and zinc finger nucleases (ZFN) are being investigated in clinical trials.
Whether the delivery of genetic material to cells occurs in vivo or in vitro, some type of vehicle or delivery aid is required to ensure that the DNA reaches its target. Both non-viral and viral methods have been employed, with the latter most widely used at the current time.
With respect to plasmids for viral-vector production, the plasmid engineering service is ideal for the optimisation of plasmids used in both AAV and LV vector manufacturing. For instance, tailor-made transgene plasmids containing cargoes up to 4.8 Kb for rAAV vectors and 8-9 kb for LV vectors can be readily generated using validated DNA. Optimum versions of the other types of plasmids required for AAV and LV vector production, such as Rep/Cap and helper plasmids, can also be engineered. All of the plasmids are suitable in current standard transfection protocols.
DNA Plasmids for Transfection
The viral vectors used for gene delivery are generally produced via transfection of several plasmid DNA molecules, one of which contains the gene of interest. The actual number depends on the type of viral vector being produced. Adeno-associated viral (AAV) vectors, which are widely used for in vivo gene therapies, typically require three plasmids, while lentiviral (LV) vectors, which are most often used for the production of CAR T-cell therapies, require four.
The specific requirements of each plasmid DNA depend on the type of viral vector and the gene of interest to be delivered. Indeed, the structure and properties of each plasmid DNA can have a direct impact on not only the transfection yield, but also on the quality of the viral particles produced.
Fine-tuning transfection for AAV manufacturing
A Growing Portfolio
The plasmid DNA engineering services complements Polyplus’ existing portfolio of nucleic acid transfection solutions for Cell and Gene therapy, biologics manufacturing and life science research. The streamlined integration and optimization of the plasmid DNA engineering services for global customers reflects a critical focus on optimizing upstream process economics for viral-vector manufacturing at Polyplus.
To inquire about the plasmid engineering service, You can email us directly at email@example.com or fill out the below form for our plasmid specialist to contact you.