Manufacture of plasmid DNA

After lysis is complete, the next step can entail the addition of a high-salt neutralization solution, such as potassium acetate. This promotes formation of aggregates of genomic DNA and 

CH14 NUCLEIC-ACID- AND CELL-BASED THERAPEUTICS 

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From the economic point of view, procedures for the manufacture of therapeutic DNA must be scalable and efficient, and, at the same time, simple and robust. Manufacturing processes are as manifold as the gene transfer methods used in gene therapy. As an example, manufacture of plasmid DNA for naked nucleic acid transfer can be briefly described as follows [26]. The methods available for plasmid production today largely originate from lab procedures for the production of DNA for analytical... 

Figure 14.11 Overview of the manufacturing process for the Large-scale production of plasmid DNA. Refer to the text for further details...

Add 1 pi of the respective siRNA stock solution (20 pM in RNA dilution buffer as supplied by the manufacturer) to each well. For plasmid transfections 1 pi of plasmid DNA at a stock concentration of 500 ng///l is added. 

Isolate plasmid DNA from recombinant E. coli cultures following the procedure recommended by the manufacturer of the maxi prep kit. Concentrate the extracted DNA by alcohol precipitation. Alternatively use Nucleobond Finalizers (contained in the NucleoBond Xtra Maxi PLUS— follow the manufacturer s protocol). DNA should be concentrated at I pg/pL, ready for downstream applications. Prior to use, verify size and quality of plasmid DNA by agarose gel electrophoresis. 

The K2 Transfection System is optimized for cell lines and promises best results during the exponential growth phase of the cells. Isolated neurons from P6-P7 mice are post-mitotic and this minimizes the uptake of DNA into the nucleus. However, with the following modifications to the manufacturer s protocol, it is possible to obtain a good expression of plasmid DNAs ... 

IEC is also important in large-scale manufacturing of gene vectors such as plasmid DNA. In one process flow scheme, IEC and size exclusion chromatography (SEC) were used sequentially to purify the plasmid after lysis... 

Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use Points to Consider on Plasmid DNA Vaccines for Preventive Infectious Disease Indications Points to Consider in the Manufacture and Testing of Therapeutic Products for Human Use Derived from Transgenic Animals... 

The issue was one of getting the DNA into a cell and this gave rise to the use of viral delivery systems and bacterial plasmid DNA (pDNA) vectors. pDNA vectors are useful since they are much safer to manufacture on a large scale, inexpensive, and easily customized—apart from being safer for the patient. However, the down side is that these systems are poorly immunogenic although, as noted above, this might be improved by the addition of an appropriate adjuvant. 

Prepare 50 pL of PCR reaction mixture according to the manufacturer s instructions (Takara) by mixing 100 pg/pL of plasmid and 200 nM of each primer with 200 pM of each dNTP and 1.25 U of ExTaq DNA polymerase. 

On the other hand, from the technological point of view, several barriers have already been overcome, allowing production of viral or plasmid DNA vectors at a cost that enables clinical application. Safe manufacturing processes are already available, and the production capacity for these systems is easily accessible worldwide. 

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