[Suppression of nonsense mutations in the Dystrophin gene by a suppressor tRNA gene]. / Ispol'zovanie gena supressornoi tRNK dlia ispravleniia nonsens-mutatsii v gene distrofina.

Nonsense mutations in the dystrophin gene are the cause of Duchenne muscular dystrophy (DMD) in 10-15% of patients. In such an event, one approach to gene therapy for DMD is the use of suppressor tRNAs to overcome the premature termination of translation of the mutant mRNA. We have carried out cotransfection of the HeLa cell culture with constructs containing a suptRNA gene (pcDNA3suptRNA) and a marker LacZ gene (pNTLacZhis) using their polymer VSST-525 complexes. It was found that the number of cells producing beta-galactosidase depends inversely on the dose of the suptRNA gene. A single in vivo injection of the construct providing for expression of the suptRNAochre gene into mdx mouse muscle resulted in the production of dystrophin in 2.5% of fibers. This suggests that suppressor tRNAs are applicable in gene therapy for hereditary diseases caused by nonsense mutations.

[Expression in vivo of the lacZ gene, delivered to mouse lungs using synthetic microspheres]. / Ekspressiia in vivo gena lacZ, dostavlennogo v legkie myshei pri pomoshchi sinteticheskikh mikrosfer.

A capacity of MF-2 synthetic microspheres to serve as the vehicle for transfer of the marker LacZ gene to mouse lung epithelial cells was studied after a single intranasal administration of the MF-2/gene complex. Two types of plasmids carrying marker gene LacZ were used in the experiments: with cytoplasmic (pCMV-LacZ) and nuclear (pCMV-nlsLacZ) localization of the gene product (beta-galactosidase). As early as 7 days after the complexes MF-2/pCMV-LacZ and MF-2/pCMV-nlsLacZ were administered, specific staining for beta-galactosidase revealed this enzyme activity in the epithelial cells of bronchi, bronchioli, and alveoli. The maximum in vivo of the marker gene in the MF-2/pCMV-LacZ complex was observed at day 14 to 21 after administration and the corresponding gene product was detected during the following two months. The MF-2-mediated gene transfer led to a twofold increase in beta-galactosidase activity relative to the case when the "unbound" pCMV-LacZ plasmid was administered. These results suggest that the synthetic microsphere-mediated transfer of alien genes to the lung of experimental animals is promising. Microspheres may be used in gene therapy for pulmonary affections, in particular cystic fibrosis.