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Updated: Jun 8, 2026
Electroporation of Plasmid DNA into Mouse Skeletal Muscle
Published on: April 6, 2022
Muscle damage after delivery of naked plasmid DNA into skeletal muscles is batch dependent
Christine I Wooddell1, Vladimir M Subbotin, Magdolna G Sebestyén
1Roche Madison, 465 Science Drive, Madison, WI 53711, USA. chris.wooddell@roche.com
Abstract:
Various plasmids were delivered into rodent limb muscles by hydrodynamic limb vein (HLV) injection of naked plasmid DNA (pDNA). Some of the pDNA preparations caused significant muscle necrosis and associated muscle regeneration 3 to 4 days after the injection whereas others caused no muscle damage. Occurrence of muscle damage was independent of plasmid sequence, size, and encoded genes. It was batch dependent and correlated with the quantity of bacterial genomic DNA (gDNA) that copurified with the pDNA. To determine whether such an effect was due to bacterial DNA or simply to fragmented DNA, mice were treated by HLV injection with sheared bacterial or murine gDNA. As little as 20 μg of the large fragments of bacterial gDNA caused muscle damage that morphologically resembled damage caused by the toxic pDNA preparations, whereas murine gDNA caused no damage even at a 10-fold higher dose. Toxicity from the bacterial gDNA was not due to endotoxin and was eliminated by DNase digestion. We conclude that pDNA itself does not cause muscle damage and that purification methods for the preparation of therapeutic pDNA should be optimized for removal of bacterial gDNA.
Insights
Bacterial genomic DNA (gDNA) contamination in plasmid DNA (pDNA) preparations causes muscle damage after hydrodynamic limb vein (HLV) injection, not the pDNA itself. Optimized purification to remove bacterial gDNA is crucial for safe therapeutic applications.
Area of Science:
- Molecular Biology
- Toxicology
- Biotechnology
Background:
- Hydrodynamic limb vein (HLV) injection is used to deliver naked plasmid DNA (pDNA) into rodent muscles.
- Some pDNA preparations cause significant muscle necrosis and regeneration, while others do not, independent of plasmid sequence, size, or gene content.
Purpose of the Study:
- To investigate the cause of muscle damage observed after hydrodynamic limb vein (HLV) injection of plasmid DNA (pDNA).
- To determine if bacterial genomic DNA (gDNA) or fragmented DNA is responsible for the observed toxicity.
Main Methods:
- Rodent limb muscles were injected with naked plasmid DNA (pDNA) via hydrodynamic limb vein (HLV) injection.
- Mice were treated with sheared bacterial or murine genomic DNA (gDNA) via HLV injection to assess toxicity.
- Toxicity was evaluated morphologically and assessed for endotoxin contamination and sensitivity to DNase digestion.
Main Results:
- Muscle damage was batch-dependent and correlated with copurified bacterial genomic DNA (gDNA) quantity.
- Bacterial gDNA fragments, but not murine gDNA, caused muscle damage morphologically similar to toxic pDNA preparations.
- Toxicity was not due to endotoxin and was eliminated by DNase digestion, indicating DNA-specific effects.
Conclusions:
- Plasmid DNA (pDNA) itself does not induce muscle damage following hydrodynamic limb vein (HLV) injection.
- Bacterial genomic DNA (gDNA) contamination is the primary cause of observed muscle necrosis and regeneration.
- Therapeutic pDNA preparation methods must be optimized to remove bacterial gDNA for safe clinical use.
