RNA interference is an evolutionarily conserved surveillance mechanism that responds to double-stranded RNA by sequence-specific silencing of homologous genes. Here we show that transgene expression can be suppressed in adult mice by synthetic small interfering RNAs and by small-hairpin RNAs transcribed in vivo from DNA templates. We also show the therapeutic potential of this technique by demonstrating effective targeting of a sequence from hepatitis C virus by RNA interference in vivo.

A major obstacle limiting gene therapy for diseases of the heart and skeletal muscles is an inability to deliver genes systemically to muscles of an adult organism. Systemic gene transfer to striated muscles is hampered by the vascular endothelium, which represents a barrier to distribution of vectors via the circulation. Here we show the first evidence of widespread transduction of both cardiac and skeletal muscles in an adult mammal, after a single intravenous administration of recombinant adenoassociated virus pseudotype 6 vectors. The inclusion of vascular endothelium growth factor/vascular permeability factor, to achieve acute permeabilization of the peripheral microvasculature, enhanced tissue transduction at lower vector doses. This technique enabled widespread muscle-specific expression of a functional micro-dystrophin in the skeletal muscles of dystrophin-deficient mdx mice, which model Duchenne muscular dystrophy. We propose that these methods may be applicable for systemic delivery of a wide variety of genes to the striated muscles of adult mammals.Human mortality and quality of life are significantly affected by diseases of the striated musculature. Genetic treatments that are being developed for conditions such as heart disease, aging-associated muscle wasting and the muscular dystrophies have been limited by an inability to achieve widespread and efficient gene transfer to the heart and dispersed skeletal muscles of an adult organism 1-4 . For example, anesthesia, invasive surgery and hazardous cofactors are required to transduce varying fractions of the cardiomyocyte population efficiently 1,5 . Similarly, the transfer of genes to the muscles of individual limbs using various vectors requires either direct injection of individual muscles, or complex surgical procedures performed under anesthesia to distribute vectors via the circulation 2-4,6-10 . Here we describe a simple and highly efficient method to transfer genes systemically to the cardiac and skeletal muscles of adult mammals. This approach uses intravenous administration of recombinant adeno-associated virus pseudotype 6 (rAAV6-pseudotyped vectors) 11 , which are extremely effective at transducing skeletal muscles after intramuscular injection 12 .Correspondence should be addressed to J.S.C. (jsc5@u.washington.edu).. 4 These authors contributed equally to this work.Note: Supplementary information is available on the Nature Medicine website. COMPETING INTERESTS STATEMENTThe authors declare competing financial interests (see the Nature Medicine website for details). NIH Public Access RESULTS Systemic transduction of skeletal muscles by rAAV6First, we examined the potential for systemic gene transfer after intravenous administration of rAAV6 vectors at the whole-body level in young adult (6-8 wk) C57Bl/10J mice (Fig. 1). The muscles of mice examined 11 days after administration through the tail vein of ~2 × 10 11 vector genomes of rAAV6 vector containing a CMV-lacZ expression cassette, did not show obvious exogenous β-galactosidase (...

The development of non-viral gene-transfer technologies that can support stable chromosomal integration and persistent gene expression in vivo is desirable. Here we describe the successful use of transposon technology for the nonhomologous insertion of foreign genes into the genomes of adult mammals using naked DNA. We show that the Sleeping Beauty transposase can efficiently insert transposon DNA into the mouse genome in approximately 5-6% of transfected mouse liver cells. Chromosomal transposition resulted in long-term expression (>5 months) of human blood coagulation factor IX at levels that were therapeutic in a mouse model of haemophilia B. Our results establish DNA-mediated transposition as a new genetic tool for mammals, and provide new strategies to improve existing non-viral and viral vectors for human gene therapy applications.