Systemic delivery of genes to striated muscles using adeno-associated viral vectors

Abstract

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 (...

Sonoporation using microbubble BR14 promotes pDNA/siRNA transduction to murine heart

“…Administration with the vascular endothelial growth factor (VEGF) may facilitate transfer of genetic materials through the endothelium, due to an increase in permeability of the endothelial cell membrane [43]. ECG-triggered sonication may also be effective because ultrasound-based cavitation can be generated precisely at the diastolic phase, when the coronary blood flow is higher than in the systolic phase.…”

Heart-targeted adeno-associated viral vectors selected by in vivo biopanning of a random viral display peptide library

“…40 Tissue directed in vivo gene transfer has also been attempted by systemic application of pseudotyped AAV6, AAV8 or AAV9 vectors. 38,39,[42][43][44][45] Besides the fact that there is only a limited number of vector serotypes available, by far lower than the variety of potential tissue targets, these serotype-based targeted vectors also have limited or no specificity for the tissue of interest. In congruence with this assumption, our data showed that the library-derived vectors presented here were more specific than AAV serotype 9 (at least with respect to biodistribution of the genomes), which is known to confer efficient transduction of heart tissue.…”

Stable transduction of myogenic cells with lentiviral vectors expressing a minidystrophin

“…Most of these immune responses could be blunted by the use of modified vector backbones (eg by using 'gutted' vectors), muscle-specific promoters (in either Ad or rAAV vectors), or by using ultrapurified vector preparations (eg HPLC purification of lentiviral vectors). 5,6,23,24,43 However, we observed that muscles transduced with the L-CK6-ntsLacZ still showed decreased b-galactosidase activity with increasing time (data not shown), while dystrophin expression from the L-CMV-DH2-R19 vector appeared stable (Figure 2b).…”

“…A 571-bp CK6 regulatory cassette derived from the mouse muscle creatine kinase (MCK) gene has previously been shown to maintain muscle-specific activity when tested in Ad and rAAV vectors, which exist as episomes in host cells. 6,23,25 However, since lentiviral vectors integrate into the host genome, we asked whether sequences within the vector genome might interfere with the muscle and differentiation specific activity of the CK6 promoter.…”

“…3,4 Also, delivery of full-length and some truncated dystrophins to adult skeletal muscles using viral vectors has been shown to reverse partially the dystrophic pathology in mdx limb muscles. 2,[4][5][6] Successful gene and cell therapy for DMD will require sustained dystrophin expression in skeletal and cardiac muscles. 2 To date, most efforts toward dystrophin delivery have used adenoviral (Ad) or recombinant adeno-associated viral (rAAV) vectors, neither of which has been shown to integrate into myonuclear genomic DNA.…”