RNA and DNA expression vectors containing genes for chloramphenicol acetyltransferase, luciferase, and beta-galactosidase were separately injected into mouse skeletal muscle in vivo. Protein expression was readily detected in all cases, and no special delivery system was required for these effects. The extent of expression from both the RNA and DNA constructs was comparable to that obtained from fibroblasts transfected in vitro under optimal conditions. In situ cytochemical staining for beta-galactosidase activity was localized to muscle cells following injection of the beta-galactosidase DNA vector. After injection of the DNA luciferase expression vector, luciferase activity was present in the muscle for at least 2 months.
Plasmid pRSVL persisted and expressed luciferase for at least 19 months in mouse skeletal muscle after intramuscular injection. Other injected plasmids also stably expressed long-term suggesting that any plasmid DNA could stably persist and express in muscle. Plasmid DNA was demonstrated by quantitative PCR in some of the muscle DNA samples for at least 19 months after injection. The methylation pattern of the plasmid DNA remained in its bacterial form indicating that the foreign DNA did not replicate in the muscle cells. The electroporation of total cellular DNA from injected muscles into bacteria indicated that the plasmid DNA was extrachromosomal. Chromosomal integration of plasmid DNA was searched for by electroporating the injected muscle DNA into bacteria after restriction enzyme digestion and ligation. No plasmids containing plasmid/chromosome junctions were observed in over 1800 colonies examined. Lack of integration increases the theoretical safety of this gene transfer technique. Long-term stability of plasmid DNA in muscle indicates that muscle is an attractive target tissue for the introduction of extrachromosomal plasmid or viral DNA for the purpose of gene therapy.
Duchenne's muscular dystrophy (DMD), which affects one in 3,500 males, causes progressive myopathy of skeletal and cardiac muscles and premature death. One approach to treatment would be to introduce the normal dystrophin gene into diseased muscle cells. When pure plasmid DNA is injected into rodent skeletal or cardiac muscle, the cells express reporter genes. We now show that a 12-kilobase full-length human dystrophin complementary DNA gene and a 6.3-kilobase Becker-like gene can be expressed in cultured cells and in vivo. When the human dystrophin expression plasmids are injected intramuscularly into dystrophin-deficient mdx mice, the human dystrophin proteins are present in the cytoplasm and sarcolemma of approximately 1% of the myofibres. Myofibres expressing human dystrophin contain an increased proportion of peripheral nuclei. The results indicate that transfer of the dystrophin gene into the myofibres of DMD patients could be beneficial, but a larger number of genetically modified myofibres will be necessary for clinical efficacy.
High titre (10(11)-10(12) pfu/ml) suspensions of autonomously replication-defective type 5 human adenovirus (AV) recombinants with different reporter gene inserts (CMV-Luciferase (Lux), CMV-beta-galactosidase (Lac Z), RSV-Lux and RSV-Lac Z) were injected into intact quadriceps muscles of 1-5 day old (Group 1) or 35-45 day old (Group 2) normal mice, as well as regenerating adult mouse muscles (Group 3) and 35 day old mdx muscles (Group 4). The expression of the reporter genes was quantitated 10 days and 2 months later. At 10 days postinjection all reporter gene expression was very high in the neonatally injected (Group 1) muscles. In Group 2 muscles the transduction was markedly less. In Group 3 muscles the gene expression was significantly better than in the Group 2 muscles. In adult mdx muscles (Group 4) where spontaneous regeneration is usually present, the results were similar to those in Group 3 animals. At 2 months post-injection in Group 1 animals, the RSV-Lux expression was even higher than at 10 days postinjection. The cell surface density of alpha v-integrin-containing molecules including the internalization receptor for AV in Groups 1, 2, 3 and 4 showed a positive correlation with AV transducibility. We conclude that adenovirus vector in high titre (10(10) pfu/ml or above) is capable of efficiently transducing only immature muscle cells but not mature muscle fibers in vivo and this appears to correlate with a higher surface density of the available AV internalization receptor in immature muscle cells and lower level in mature muscle fibers.(ABSTRACT TRUNCATED AT 250 WORDS)
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