Background High levels of expression in hepatocytes can be achieved after intraportal delivery of plasmid DNA vectors with up to 10% of all liver cells transfected. CMV promoter-driven expression is very high on Day 1 after injection, but is diminished strongly by Day 2. Expression slowly declines after 1 week. We describe experiments aimed at elucidating the reasons for this rapid decline in transgene expression.Methods Histological methods were used to determine the presence and extent of Liver damage and hepatocyte proliferation. Viral and liver-specific promoters were tested to study promoter shut-off, Southern blotting was performed to determine the loss of the pDNA vector over time, and several mouse models were used to study the host immunological response.Results pDNA is lost rapidly early after injection, but remains at a relatively stable copy number after Day 4. Southern blotting experiments showed that plasmid DNA could be detected for at least 12 weeks after injection (0.2 copies per genome). The early rapid decline of expression is promoter dependent. A liver-specific albumin promoter resulted in similar levels of expression on Days 1 and 7, suggesting that promoter inactivation may be responsible for the instability of CMV promoter-driven expression. The slow decline in expression levels after 1 week appears to be the result of an immune response directed against the expressed transgene. Expression was much prolonged in immunosuppressed, immunodeficient, or antigen-tolerized mice.Conclusion The present data suggest that if promoter inactivation can be overcame, intravascular delivery of plasmid DNA could be a highly efficient, simple and non-toxic liver gene therapy approach. Intravascular delivery of pDNA allows for the rapid screening of novel expression vectors in vivo. Copyright (C) 2001 John Wiley & Sons, Ltd.

Previous studies have demonstrated that muscle can take up and express naked DNA or RNA. This study demonstrates that the pDNA can be delivered to and expressed within skeletal muscle when injected rapidly, in a large volume and when all blood vessels leading into and out of the hindlimb are occluded. The additional use of collagenase, papaverine and ischemia raised expression moderately but was not critical. These results demonstrate that a nonviral method can lead to high levels of expression in the muscles of adult animals larger than mice.

There is a time window at 2 weeks of age for achieving very high levels of foreign gene expression from the intramuscular injection of naked plasmid DNA in mice and rats. The highest expression, over 1 microg of luciferase protein/muscle, was obtained in Balb/C mice using constructs containing the CMV promoter, a chimeric intron and the luc+ luciferase gene. Approximately 50% of the myofibers were intensely blue following the intramuscular injection of a beta-galactosidase expression vector in 2 week old Balb/C mice. The effects of age, mouse strain and construct were multiplicative, resulting in >1000-fold greater luciferase and approximately 20-fold more beta-galactosidase-positive cells. These high levels of expression were unstable and were not observed in larger animals (dog, rhesus monkey). These results indicate that enormous levels of foreign gene expression can be obtained in muscle with naked DNA in vivo and will enable the temporary effects of gene function and expression in rodent muscle to be expeditiously studied.

Sindbis virus was used as a self-amplifying eukaryotic expression vector. A recombinant cDNA genome of this (+)-strand RNA virus was placed under the transcriptional control of a Rous sarcoma virus LTR (RSV) promoter. Transfection of this plasmid construct into mammalian cell lines (3T3, HepG2, and 293 cells) resulted in expression of the luciferase reporter gene. High-expression levels were also measured after transfection into primary rat myoblasts. In differentiated myotubes, expression levels generated by the Sindbis virus vector were up to 200 times higher than those obtained with a conventional RSV expression vector. In vivo expression was detected after injection of plasmid DNA into mouse quadriceps. In vivo expression was transient and undetectable by day 16. This self-amplifying expression vector can be used for generating high-level expression of transgenes in vitro and in vivo. Its transient nature in vivo could allow for safe, short-term delivery of gene products in gene therapy protocols. It should facilitate the study of Sindbis and other RNA viruses.

These studies were initiated to elucidate the mechanism of DNA nuclear transport in mammalian cells. Biotin- or gold-labeled plasmid and plasmid DNA expression vectors for Escherichia coli beta-galactosidase or firefly luciferase were microinjected into the cytoplasm of primary rat myotubes in culture. Plasmid DNA was expressed in up to 70% of the injected myotubes, which indicates that it entered intact, postmitotic nuclei. The nuclear transport of plasmid DNA occurred through the nuclear pore by a process common to other large karyophilic macromolecules. The majority of the injected plasmid DNA was sequestered by cytoplasmic elements. This understanding of plasmid DNA nuclear transport provides a basis for increasing the efficiency of gene transfer.

We have previously proposed the use of primary muscle cells as a “platform,” or “vehicle” for intracerebral transgene expression. Brain grafts of minced muscle, or cultured muscle cells persisted in rat brains for at least 6 mo without any decrease in graft size, or tumor formation. Stable, but moderate levels of intracerebral transgene expression were obtained by transplanting plasmid-transfected myotubes in culture. In the present study, high and stable levels of intracerebral transgene expression were achieved by the co-transplantation of plasmid-transfected myoblasts and myotubes in culture. Approximately 5 × 10s myoblasts and myotubes were transfected with 10 μg pRSVL plasmid DNA, and 30 μg Lipofectin (BRL), respectively. They were mixed together (total cell number was 1 million), and stereotactically injected into the caudate nucleus of an adult rat brain. The activity of luciferase, the product of transgene expression, was stable for at least 4 mo, and much higher than the levels in myotube grafts, or co-grafts of myoblasts and minced muscle. Presumably, the myotubes served as a framework on which the myoblasts can form myotubes. The sections of brains transplanted with co-graft of myoblasts, and myotubes transfected with pRSVLac-Z were stained immunofluorescently for β-galactosidase activity. The muscle grafts contained β-galactosidase positive myofibers 4 mo after transplantation. Such high and stable levels of in vivo expression after postnatal gene transfer have rarely been achieved. Primary muscle cells are useful vehicle for transgene expression in brains, and potentially valuable for gene therapy of degenerative neurological disorders.

Previously, we showed that rodent muscle has the ability to take up and express plasmid genes injected intramuscularly. This study now demonstrates that nonhuman primate muscle also has this ability to express injected plasmids. A scaled-up version of the standard large preparation of plasmid DNA allowed several tens of milligrams of CCC plasmid DNA to be relatively easily produced and administered to monkeys. After the injection of the E. coli beta-galactosidase reporter gene in pRSVLac-Z, foreign gene expression was localized to both type I and type II myofibers. The luciferase reporter gene in pRSVL was used to quantify the amount of expression. The multiple implantation of plasmid DNA pellets was more efficient in expressing luciferase than the injection of DNA in normal saline. Luciferase activity persisted for at least 4 months after injection. However, the luciferase expression was considerably less than that in rodents. Preliminary studies explored why expression was less in monkeys. Of particular interest was the increased thickness of the perimysium of monkeys as compared to that in rodents. This increased connective tissue may decrease delivery of the plasmid DNA to the myofibers. Anti-nuclear or anti-DNA antibodies were not observed, even after repetitive DNA administrations, and no adverse effects were observed in any of the monkeys.

This report extends our previous findings that mouse muscle cells in situ can take up naked DNA injected intramuscularly in vivo. Various conditions such as needle type, speed of injection, volume of injection fluid, tonicity of injection fluid, type of solute, type of muscle, physiologic condition of the muscle and age of the animals were appraised for their effect on the levels of luciferase activity expressed from the pRSVL plasmid. Specific conditions such as the use of normal saline as an injection fluid increased the efficiency of expression. The implantation of DNA pellets was an effective way to deliver DNA to muscle, especially for smaller muscle groups. Also, newborn and adult rat muscles expressed plasmid DNA delivered intramuscularly.