No abstract

Adenoviral vector-mediated gene transfer to skeletal muscle is a promising potential treatment for Duchenne muscular dystrophy. However, the immunological response to viral antigens and the therapeutic protein expressed by the delivered gene could prevent effective treatment. In this study, we investigated the immune response induced by adenoviral and dystrophin antigens presented by high-capacity adenoviral vector-mediated dystrophin and beta-galactosidase delivery to skeletal muscle of a mouse model that is both dystrophin-deficient and lacZ transgenic. Direct intramuscular gene delivery of the high-capacity adenoviral vector encoding full-length murine dystrophin resulted in stable expression of recombinant dystrophin for 5 months in mice treated as neonates and for 4 weeks in mice treated as adults. We observed neutralizing antibody to adenoviral antigens only in mice treated as adults and not in mice treated as neonates. This suggested that adenoviral antigens were only presented at the time of vector administration when the neonatal immune system was not yet mature. In contrast, antibodies to dystrophin were observed both in mice treated as neonates and in mice treated as adults. The development of an anti-dystrophin antibody response in mice treated with the high-capacity adenoviral vector as neonates suggested that dystrophin antigens were presented to the immune system at a time remote from the gene delivery, when the immune system was mature. Interestingly, an antibody response against beta-galactosidase developed late in the course of mice treated with the high-capacity adenoviral vector as neonates, suggesting a loss of tolerance to beta-galactosidase, a self-antigen in these transgenic mice. Our results suggest that future human trials of dystrophin gene delivery will need to address the potential for immunity induced by ongoing segmental degeneration of partially treated muscle fibers and presentation of recombinant dystrophin antigens in the context of a Duchenne muscular dystrophy patient.

Duchenne muscular dystrophy (DMD) is an X-linked associated proteins. A greater amount of dystrophin recessive muscle disease characterized by a lack of dysreplacement occurred in mdx muscle following transplantrophin expression. Myoblast transplantation and gene tation of mdx myoblasts isolated from a transgenic mouse therapy have the potential of restoring dystrophin, thus overexpressing dystrophin suggesting that engineering decreasing the muscle weakness associated with this disautologous myoblasts to express high amounts of dystroease. In this study we present data on the myoblast phin might be beneficial. The ex vivo approach possesses mediated ex vivo gene transfer of full-length dystrophin to attributes that make it useful for gene transfer to skeletal mdx (dystrophin deficient) mouse muscle as a model for muscle including: (1) creating a reservoir of myoblasts capautologous myoblast transfer. Both isogenic primary mdx able of regenerating and restoring dystrophin to dystrophic myoblasts and an immortalized mdx cell line were transmuscle; and (2) achieving a higher level of gene transfer duced with an adenoviral vector that has all viral coding to dystrophic muscle compared with adenovirus-mediated sequences deleted and encodes ␤-galactosidase and fulldirect gene delivery. However, as observed in direct gene length dystrophin. Subsequently, these transduced myotransfer studies, the ex vivo approach also triggers a cellublasts were injected into dystrophic mdx muscle, where the lar immune response which limits the duration of transinjected cells restored dystrophin, as well as dystrophingene expression.

To determine the role of the nerve on the establishment of myofiber diversity in skeletal muscles, the lumbosacral spinal cord of 14‐day gestation mice (E14) was laser ablated, and the accumulation of the myosin alkali light chains (MLC) mRNAs in crural (hindleg) muscles was evaluated just prior to birth with in situ hybridization. Numbers of molecules of each alkali MLC/ng total RNA in the extensor digitorum longus (EDL) and soleus muscles were determined with competitive polymerase chain reaction. Transcripts for all four alkali MLCs accumulate in aneural muscles. Data suggest that: (1) the absence of the nerve to either future fast or slow muscles results in less accumulation of MLC1V transcript. Moreover, the presence of the nerve is required for the enhanced accumulation of this transcript in future slow muscles; (2) the absence of innervation of future slow, but not fast, muscles decreases the accumulation of MLC1A transcript. Since increased accumulation of MLC1A and MLC1V transcripts are found in future slow muscles at birth, the nerve is necessary for the development of the slow phenotype during myogenesis; (3) MLC1F and MLC3F transcripts do not display any preferential accumulation in future fast muscles during the fetal period. Therefore, the establishment of the differential distribution of these mRNAs, based on fiber type, is a postnatal phenomenon. The nerve is required during the fetal period to allow accumulation of MLC3F messages above a basal level in future fast as well as slow muscles; whereas, the absence of the innervation to future fast, but not slow, muscles reduces the accumulation of MLC1F. Thus, the accumulation of the various alkali MLC mRNAs shows a differential, rather than coordinate, response to the absence of the nerve, and this response may vary depending on the future fiber type of the muscles. Dev. Dyn. 1998;211:177–190. © 1998 Wiley‐Liss, Inc.

The postnatal elimination of embryonic and neonatal myosin isoforms in the rat extensor digitorum longus, diaphragm, and soleus muscles was compared using electrophoresis and immunohistochemical techniques. Electrophoresis of native myosin showed that neonatal bands were present in all three muscles on day 4 but were absent from the day 21 extensor digitorum longus muscle that exhibited its adult electrophoretic pattern. Mature electrophoretic banding patterns were present on days 60 and 125 in the diaphragm and soleus muscles, respectively. Immunohistochemical analysis indicated that embryonic myosin heavy chain was present in all day 4 samples but absent by day 21. Quantitative evaluation determined that the rate of elimination of neonatal myosin heavy chain (MHCneo) was faster in the extensor digitorum longus muscle than in the diaphragm, with the soleus muscle having the slowest rate of elimination of this isoform. Embryonic myosin light chain was detected by two-dimensional electrophoresis through day 8 in each of the muscles. These data indicate that postnatal elimination of MHCneo is tissue specific and time dependent but not governed by either activity level or rostral-caudal position.