Features of transfection and expression of human dystrophin cDNA deliveredinto the MDX mouse muscle by means of MF-2 synthetic microspheres

The number of dystrophin-positive myofibers (DPM) that appeared in differen t skeletal muscles of mdx mice after a single injection of synthetic micros pheres containing the full-length human dystrophin cDNA within the pHSADy e xpressing vector into femoral quadriceps muscle was examined on cryostat se ctions. Injection of 25 mu g cDNA resulted in the occurrence of 1, 2.4, 5.8 , and 4.8% of DPM in the treated muscle in I, 7, 21, and 60 days after the injection respectively. 7, 21, and 60 days after the treatment, these value s comprised 2.1, 4.3 and 1% in the same muscle of the contralateral leg, an d 5.5, 8.4, and 1% in the gluteal muscle. Expression of the full-length hum an dystrophin (427 kDa) in the muscle of the transfected mdx mice was obser ved. The presence of the transfected construction in skeletal muscles, hear t, brain, lung, and fetuses was demonstrated by PCR Utilization of the FISK technique with biotinilated pHSADy construct as a DNA probe showed that 7 days after injection, the MF-2 microspheres were present in 70% of myoblast nuclei, in 64% of nuclei of gluteal muscles, and in 62% of the contralater al quadriceps nuclei. 21 days after the treatment, these values were 41, 29 , and 45%, respectively The MF-2 microspheres were detected in the nuclei o f the blood, brain, heart, and lung cells, as well as in the placenta and t issues 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 f or at least two months after injection. The MF-2 microspheres attract speci al interest in respect to the targeted delivery of gene constructs into the nuclei.