Local and distant transfection of mdx muscle fibers with dystrophin and LacZ genes delivered in vivo by synthetic microspheres

Abstract

Patterns of dystrophin and ␤-galactosidase expression ticles were detected by FISH analysis in about 60-70% of were examined in mdx mice after i.m. injections of synmyofiber nuclei in muscles of injected and contralateral thetic microspheres (MF-2) loaded with full-length limbs 7 days after application. The presence of human dys-(pHSADy) or mini-dystrophin gene (pSG5dys) cDNA plastrophin cDNA and its products in all skeletal muscles and mid constructs or with LacZ marker gene (pCMV-LacZ). A in different internal organs was proven by PCR and RTsingle injection of 25 g pHSADy into quadriceps femoris PCR analysis. Patches of ␤-galactosidase expression were muscle resulted in 6.8% of dystrophin positive myofibers abundant in injected muscle, and frequent in the contralat-(DPM) in a given muscle; 8.4% of DPM in glutaeus muscle eral and other skeletal muscles as well as in diaphragm, and 4.3% of DPM in quadriceps femoris muscle of contraheart and lungs. High levels of dystrophin cDNA lateral limb on day 21 after exposure compared with only expression, and an efficient distant transfection effect with 0.6% DPM in intact (non-injected) mdx mice. A high propreferential intranuclei inclusion of MF-2 vehicle, are very portion of DPM (17.6% and 10.8%, respectively) was regisencouraging for the development of a new constructive tered in both injected and contralateral muscles after ministrategy in gene therapy trials of DMD. gene cDNA administration. MF-2/dystrophin cDNA par-

Intramuscular delivery of DNA releasing microspheres: Microsphere properties and transgene expression

“…This observation suggests that sustained release formulations may enhance and prolong transgene expression without requiring multiple interventions. Microspheres and nanospheres injected intramuscularly have demonstrated the capacity of promoting and prolonging transgene expression, though the underlying mechanisms are not well understood [19][20][21][22][23][24][25][26].…”

Transfection of large plasmids in primary human myoblasts

“…The truncated dystrophin minigene has been introduced in myoblasts or myofibers using a E1-deleted adenoviral vector, 6,7 a retroviral vector 8 and MF2 particles. 9 The full-length dystrophin cDNA has been introduced in myoblasts or myofibers using a helper-Gene Therapy dependent adenovirus, 10 HVJ liposomes, 11 ballistic gene transfer 12 and MF2 particles. 9 The titer of the retrovirus containing the minidystrophin cDNA is very low and thus very few myoblasts are infected.…”

“…9 The full-length dystrophin cDNA has been introduced in myoblasts or myofibers using a helper-Gene Therapy dependent adenovirus, 10 HVJ liposomes, 11 ballistic gene transfer 12 and MF2 particles. 9 The titer of the retrovirus containing the minidystrophin cDNA is very low and thus very few myoblasts are infected. The helperdependent adenovirus is quite tedious to produce and therefore is not a versatile method to study the introduction of large DNA constructs into cells.…”

Progress in gene therapy for Duchenne muscular dystrophy

“…For example, synthetic microspheres combine DNA with biodegradable synthetic polymer. Baranov et al [57] report on the use of synthetic microspheres as a delivery vehicle to administer the dystrophin cDNA to mdx muscle fibers. The potential advantage of nonviral vector-mediated strategies would be to circumvent the immune response and transgene size limitations inherent in viral vectorbased approaches.…”