Trans-Splicing Adeno-Associated Viral Vector-Mediated Gene Therapy Is Limited by the Accumulation of Spliced mRNA but Not by Dual Vector Coinfection Efficiency

Abstract

Therapeutic application of recombinant adeno-associated virus (AAV) has been limited by its small carrying capacity. To overcome this limitation trans-splicing vectors were developed recently. However, the transduction efficiency of trans-splicing vectors is considerably lower than that of a single intact vector in skeletal muscle. To improve trans-splicing vectors for skeletal muscle gene therapy, we examined whether coinfection efficiency is a rate-limiting factor in the mdx mouse, a model for Duchenne muscular dystrophy. Two different AAV viruses were delivered to the mdx muscle. Similar to previous reports in normal muscle, coinfection efficiency reached approximately 90% in the diseased muscle. This result suggests that coinfection is not a hurdle in dystrophic muscle. Another critical step in the trans-splicing method is the transcription and splicing across the inverted terminal repeat (ITR) junction in the reconstituted genome. To test whether this represented a significant obstacle, we systematically evaluated the transcription, pre-mRNA stability and splicing, and translation in a synthetic lacZ construct that mimicked the reconstituted genome. Although inserting an intron in the lacZ gene had no effect on its expression, inclusion of the ITR junction in the intron reduced expression by 50%. In construct containing the ITR junction, the mRNA transcript level was significantly reduced. This mRNA level reduction was associated with decreased pre-mRNA stability. These data suggest that the accumulation of mRNA is a rate-limiting factor in trans-splicing vector-mediated gene therapy.

Treatment of human disease by adeno-associated viral gene transfer

“…Co-administration of these vectors to mdx mice resulted in successful expression of the mini-dystrophin with improvements in skeletal muscle function (Lai et al 2005a). The accumulation of spliced message appears to be the major limiting factor to this strategy (Xu et al 2004).…”

Translational Research and Therapeutic Perspectives in Dysferlinopathies

“…The encapsidation size is limited to approximately 4 kb, the equivalent of three C2 domains (83). To solve this problem, researchers used the concatemerization property of AAV (84,85). Two independent AAV vectors are used: one carrying 5′ dysferlin cDNA fused with an intronic sequence that carries a donor splice site and the other containing the 3′ cDNA-bearing intronic sequence and a splice acceptor site.…”

Expanding Adeno-associated Viral Vector Capacity: A Tale of Two Vectors

“…However, the actual transduction efficiency is much lower than that of a single intact AAV vector (Duan, et al, 2001). There are a number of potential rate limiting factors in the trans-splicing approach (Lai, et al, 2005;Xu, et al, 2004;Yan, et al, 2005). These include co-infection efficiency, formation of the head-to-tail heterodimer genome, transcription and the stability of the double-D ITR containing pre-mRNA and splicing across the double-D ITR structure ( Figure 5).…”

“…It is now clear that co-infection is not a barrier. Even in a diseased tissue, co-infection efficiency can reach 90% (Xu, et al, 2004). To test whether unidirectional heterodimer formation is a rate-limiting step, Yan et al generated trans-splicing AAV vectors with AAV-2 ITR and AAV-5 ITR at the opposite ends of the genomes .…”