Gene Therapy by Intramuscular Injection of Plasmid DNA: Studies on Firefly Luciferase Gene Expression in Mice

Abstract

Direct injection of nonviral, covalently closed circular plasmid DNA into muscle results in expression of the DNA in myofiber cells. We have examined the expression of firefly luciferase DNA constructs injected into adult murine skeletal muscle. Considerable variation in luciferase enzyme expression was noted among constructs with different regulatory elements, among different batches of the same DNA construct, and among similar transfection experiments performed at different times. This variation was minimized by using single batches of plasmid DNA and by performing comparable sets of experiments concurrently. A quantitative experimental protocol was defined for comparing various aspects of the transfection process. We report that a luciferase construct containing the human cytomegalovirus immediate-early gene promoter plus intron A (a construct termed "p-CMVint-lux") showed the highest expression among several constructs tested. Dose-response and time course analyses of p-CMVint-lux DNA injections showed that maximal luciferase expression was achieved with 25 micrograms of DNA at 7-14 days post-injection. Selected manipulations of the transfection process were examined for their influence on luciferase expression. Variations in the rate of DNA injection, needle size, injection volume, and vehicle temperature had no significant effect on luciferase expression. The presence of endotoxin, cationic peptide, muscle stimulants or relaxants, vasoconstrictors, metal chelators, or lysosomal lytic reagents had no significant effect on expression. However, linearization of the DNA, injection of the DNA in water rather than saline, or inclusion of a DNA intercalating agent nearly abolished luciferase expression. And finally, increasing the injection dose by giving multiple injections over a 10-day period increased expression proportionally to the number of injections.

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.…”

Minicircle: an improved DNA molecule for in vitro and in vivo gene transfer

“…1 Production and purification of the CMV luc+ and CMV ␤-gal minicircles Bacteria were grown at 30°C in LB medium containing 50 mg/ml kanamycin. Plasmids 3186 and 3187 were used to transform E. coli strains D1210 (F − hsdS20, supE44, ara-14, galK2, proA2, leuB6, rpsL20, xyl5, mtl1, recA, mcrB, ⌬(mcrC-mrr), lacI Q ) 27 and D1210HP (D1210 lysogenized with cI857 xis − kil − phage). The phage lytic cycle was initiated in D1210HP cultures by incubation for 10 min at 42°C, which caused site-specific recombination, leading to the production of a minicircle and a miniplasmid.…”

Liposome-mediated gene transfer into human basal cell carcinoma

“…20 The cDNA for the different IFN subtypes was generated by RT-PCR using for human IFN-␣2: GCTAGGATAT CACCATGGCCTTGACCTTTGCTTTAC and ATCGGA TCCTCATTCCTTACTTCTTAAACTTTC, for human IFN-␣4 GCTAAGATATCACCATGGCCCTGTCCTTTT CTTTAC and ATCGGATCCTCAATCCTTCCTCCTTA ATCTTTTTTG, for human IFN-␣7 GCTAGGATATCAC CATGGCCCGGTCCTTTTCTTTAC and ATCGGATCC TCAATCCTTCCTCCTTAATCC, for human IFN-⍀1 GCTAGGATATCACCATGGCCCTCCTGTTCCCTCTAC and ATCGGATCCTCAAGATGAGCCCAGGTCTATA TC, for mouse IFN-␣2 ATCGGATCCTCACTCCTT CTCTTCACTCAGTC and GCTAGGATATCACCATGG CTAGACTCTGTGCTTTCC and for mouse IFN-␣7 GCTAGGATATCACCATGGCTAGGAAGGATCAGGA CC and ATCGGATCCTCACTCCTTCTTCTCACTCAG TCTTGCC and total RNA from human or mouse PBMC. The PCR products were digested with EcoRV and BamHI and ligated into the VR1332 31 vector which had been cut with the same enzymes. IRE-CAT with the CAT reporter gene under the control of the interferon responsible element has been previously described.…”