Efficient in vivo gene expression by trans-splicing adeno-associated viral vectors

Abstract

Although adeno-associated virus (AAV)-mediated gene therapy has been hindered by the small viral packaging capacity of the vector, trans-splicing AAV vectors are able to package twice the size of the vector genome. Unfortunately, the efficiency of current trans-splicing vectors is very low. Here we show that rational design of the gene splitting site has a profound influence on trans-splicing vector-mediated gene expression. Using mRNA accumulation as a guide, we generated a set of efficient trans-splicing vectors and achieved widespread expression of the 6-kb DeltaH2-R19 mini-dystrophin gene in skeletal muscle of mdx mice, a model for Duchenne muscular dystrophy. The dystrophic phenotype was ameliorated in both adult and aged mice. This demonstrates the use of trans-splicing vectors to efficiently express a large therapeutic structural protein. This strategy should be applicable to other large therapeutic genes or large transcription regulatory elements.

Immunity to adeno-associated virus vectors in animals and humans: a continued challenge

“…Trans-splicing vectors have been developed, which are able to efficiently package twice the size of the vector genome. 10 Finally, AAV gene-targeting vectors have also recently been used successfully to correct naturally occurring gene mutations through homologous recombination. 11 Rather than delivering an episomal copy of a therapeutic gene, this novel approach allows efficient, specific, nonmutagenic gene repair in vivo.…”

Systemic AAV-9 transduction in mice is influenced by animal age but not by the route of administration

“…It has been shown that AAV-2 preferentially transduces slow-twitch fibers while AAV-6 transduces both fiber types efficiently. [8][9][10] It is not clear whether AAV-9 displays a fiber type preference. To address this issue, we compared AP expression in the tibialis-anterior (TA) muscle (fast twitch predominant) and the soleus muscle (slow twitch predominant).…”

Cystic fibrosis transmembrane conductance regulator with a shortened R domain rescues the intestinal phenotype of CFTR −/− mice

“…Two studies reported that AAV vectors could package and express transgenes that substantially exceeded this limit, although at reduced efficiency (14,17). However, more recent studies have demonstrated very inefficient packaging when the expression cassette exceeded a length of ∼5 kb (16,19,38). When attempts were made to incorporate large transgenes into AAV vectors, the transgenes tended to fragment and rearrange.…”

“…An earlier comprehensive study found that both packaging and expression of cDNAs in an AAV vector were optimal when the incorporated expression cassette was less than ∼4.8 kb (15). Since then, multiple attempts have been made to design expression cassettes that fit within this limit, primarily by limiting the size of the transgene (36,37) or infecting with two vectors, each expressing half of the transgene (11,38). Two studies reported that AAV vectors could package and express transgenes that substantially exceeded this limit, although at reduced efficiency (14,17).…”