Features of transfection and expression of human dystrophin cDNA delivered into the mdx mouse muscle by means of MF-2 synthetic microspheres

摘要

The number of dystrophin-positive myofibers(DPM), that appeared in different skeletal muscles of mdx mice after a single injection of synthetic microspheres containing the full-length human dystrophin cDNA within the pHSADy expressing vector into femoral quadriceps muscle, was examined on cryostat sections. Injection of 25 #mu#g cDNA resulted in the occurrence of 1, 2.4, 5.8 and 4.8percent of DPM in the treated muscle in 1, 7, 21 and 60 days after the injection respectively, 7, 21, and 60 days after the treatment, these values comprised 2.1, 4.3 and 1percent in the same muscle of the contralateral leg, and 5.5, 8.4 and 1percent in the gluted muscle. Expression of the full-length human dystrophin in the muscle of the transfected mdx mice was observed. The presence of the transfected construction in skeletal muscles, heart, brain, lungs and fetuses was demonstrated PCR. Utilization of the FISH technique with biotinilated pHSADy construct as a DNA probe showed that 7 days after the injection, the MF-2 microspheres were present in 70percent of myoblast nuclei, in 64percent of nuclei of gluteal muscles and in 62percent of the contralateral quadriceps nuclei. 21 days after the treatment these values were 41, 29, and 45percent respectively. The MF-2 microspheres were detected in the nuclei of the blood, brain, heart and lung cells, as well as in the placenta and tissues of 18-day-old fetuses. Our results demonstrated the high efficiency of microsphere-mediated transfer of gene constructs into cell nuclei, their long-term intranuclear persistence, and the ability to direct expression for at least 2 months after injection. The MF-2 microspheres attract special interest in respect to the targeted delivery of gene constructs into the nuclei.