An Improved Plasmid DNA Expression Vector for Direct Injection into Skeletal Muscle

Abstract

In previous work, the direct injection of 50 micrograms of a plasmid DNA vector encoding firefly luciferase (VR1205) into murine quadriceps muscle produced an average of 6.5 ng of luciferase per muscle at 7 days postinjection. In this report, various elements of the VR1205 vector were modified to increase gene expression levels or to eliminate undesired viral sequences. Expression of the modified vectors was then compared to VR1205 using the intramuscular injection assay. In general, modifications to promoter, enhancer, and intronic sequences either decreased luciferase expression levels or had no effect. However, modifications to the polyadenylation and transcriptional termination sequences, plasmid backbone elements, and the luciferase gene itself each increased luciferase expression levels. The best-expressing vector, designated VR1255, contained a combination of these incrementally beneficial changes. A single intramuscular injection of 50 micrograms of VR1255 produced 300 ng of luciferase at 7 days postinjection, an expression level 46-fold higher than the VR1205 vector (or 22-fold higher, excluding modifications to the luciferase gene) and 154-fold higher than a commercially available luciferase expression vector. Thus, VR1255 represents an improved plasmid DNA vector that may be useful for gene therapy applications.

Sequence Requirements for Plasmid Nuclear Import

“…As likely candidates, two very strong in vitro and in vivo promoters were chosen: the immediate early promoter and enhancer from the human cytomegalovirus (hCMViep) and the LTR promoter from Rous sarcoma virus (RSV-LTR). Both promoters have been shown to be even stronger than the SV40 early promoter for gene expression in mouse muscles in vivo (10,11). As expected, when pOR(5171-300)was injected into the cytoplasm, it localized largely to the nucleus within eight hours as visualized by in situ hybridization (Fig.…”

“…It is somewhat surprising that the hCMViep and the RSV LTR sequences do not promote plasmid nuclear import, especially since they are two of the strongest promoters identified to date for use in cell culture and in muscle (10,11). In transfection experiments this is not surprising since most transfections are performed on subconfluent cells that are incubated for 24 to 48 hours, allowing at least one round of cell division and access to the nucleus during the transient breakdown of the nuclear envelope.…”

“…In transfection experiments this is not surprising since most transfections are performed on subconfluent cells that are incubated for 24 to 48 hours, allowing at least one round of cell division and access to the nucleus during the transient breakdown of the nuclear envelope. However, both of these promoters generate very high levels of gene expression when injected into mouse quadricep muscles that are made up of post-mitotic, terminally differentiated myotubes (10,11). Thus, these plasmids gain access to the nuclei of these cells (33).…”

Safety of a GM-CSF adjuvant-plasmid DNA malaria vaccine

“…45 The VR1055 plasmid used as a control in the GM-CSF pharmacology study is similar to VR1012 except that the bovine growth hormone 3ЈUTR/poly A sequence in VR1012 was replaced by a highly modified 3Ј UTR from rabbit ␤-globin. 46 Plasmid DNA was prepared by bacterial fermentation and purification, and quality assurance analysis was performed as previously described. 43,47 Plasmid DNA was resuspended in phosphate buffered saline vehicle (PBS, 10 mm NaH 2 PO 4 , pH 7.2, 0.9% NaCl) at the desired concentration and stored at −20°C until use.…”

Intravesical liposome-mediated interleukin-2 gene therapy in orthotopic murine bladder cancer model

“…Details regarding general structures of the novel plasmid DNA vectors used in this study have been described previously. 14,41 These plasmids commonly contained the human cytomegalovirus immediate-early promoter and enhancer plus intron A with a transcription terminator from the bovine growth hormone gene and kanamycin resistance gene for selection. VR1019 is the parental plasmid DNA expression vector.…”