Transfer of the Full-Length Dystrophin-Coding Sequence into Muscle Cells by a Dual High-Capacity Hybrid Viral Vector with Site-Specific Integration Ability

Abstract

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene, making it a potential target for gene therapy. There is, however, a scarcity of vectors that can accommodate the 14-kb DMD cDNA and permanently genetically correct muscle tissue in vivo or proliferating myogenic progenitors in vitro for use in autologous transplantation. Here, a dual high-capacity adenovirus-adeno-associated virus (hcAd/AAV) vector with two full-length human dystrophin-coding sequences flanked by AAV integration-enhancing elements is presented. These vectors are generated from input linear monomeric DNA molecules consisting of the Ad origin of replication and packaging signal followed by the recently identified AAV DNA integration efficiency element (p5IEE), the transgene(s) of interest, and the AAV inverted terminal repeat (ITR). After infection of producer cells with a helper Ad vector, the Ad DNA replication machinery, in concert with the AAV ITR-dependent dimerization, leads to the assembly of vector genomes with a tail-to-tail configuration that are efficiently amplified and packaged into Ad capsids. These dual hcAd/AAV hybrid vectors were used to express the dystrophin-coding sequence in rat cardiomyocytes in vitro and to restore dystrophin synthesis in the muscle tissues of mdx mice in vivo. Introduction into human cells of chimeric genomes, which contain a structure reminiscent of AAV proviral DNA, resulted in AAV Rep-dependent targeted DNA integration into the AAVS1 locus on chromosome 19. Dual hcAd/AAV hybrid vectors may thus be particularly useful to develop safe treatment modalities for diseases such as DMD that rely on efficient transfer and stable expression of large genes.

Adeno-associated virus integration: virus versus vector

“…AAVS1-specific integration of the reporter cassette was detected in up to 30% of the integration sites analyzed, although with the majority of vector junctions occurring within the Ad, rather than AAV, ITR elements. In another example of hybrid vector technology, Gonçalves et al 29 demonstrated that two copies of a 14-kbp dystrophin cDNA borne by a hybrid adenovirus-AAV vector could be targeted to the AAVS1 locus within human HeLa cells when the AAV Rep proteins were provided in trans from a transfected expression plasmid. A demonstration of hybrid vectormediated site-specific integration in vivo was provided by Breakefield and colleagues 30 who used a herpes simplex virus type 1-AAV hybrid amplicon vector to successfully target the integration of an ataxia-telangiectasia mutated-encoding cDNA into the AAVS1 locus of AAVS1-bearing transgenic mice.…”

Gutless adenovirus: last-generation adenovirus for gene therapy

“…110,111 Other tissues like lung, cardiac muscle, vascular tissue or dendritic cells have also been targeted with gutless adenoviruses with similar results as for the muscle or the liver. [112][113][114][115] Future considerations This is a fascinating moment for gutless adenovirus vectors: their use permits long-term expression in vivo with reduced and transient cellular immune response in Gutless adenovirus R Alba et al animal models for human diseases and, moreover, the increasing number of groups working in this field favors technological advances to escape preimmune response by developing non-human gutless adenovirus 20 or to increase stability by generating integrative gutless vectors, 116,117 or vectors with replication capacity. 118 However, their use in clinical assays is questionable since helper contamination levels are still high, and large-scale production in bioreactors is not yet fully developed.…”

New Vectors for Stable and Safe Gene Modification

“…Therefore helper dependent (HD) Ad vectors depleted of all viral genes should be used for stem cells transduction. In fact most Ad/AAV hybrid systems are based on HD Ad vectors which have demonstrated their ability to stably express several transgenes by specific integration into the AAVS1 integration site, both in vitro and in vivo achieving expression of therapeutic levels of Factor VIII in hemophilia A mice (Gnatenko et al, 2004) and of dystrophin in mdx mice (Goncalves et al, 2005). Although the Ad/AAV hybrid system has achieved specific integration into the AAVA1 site, the efficiency is far from 100%.…”