AAV Vectors Containing rDNA Homology Display Increased Chromosomal Integration and Transgene Persistence

Oregon Stem Cell Center, Papé Family Pediatric Research Institute, Oregon Health and Science University, Portland, Oregon, USA.
Molecular Therapy (Impact Factor: 6.23). 09/2012; 20(10):1902-11. DOI: 10.1038/mt.2012.157
Source: PubMed


Although recombinant adeno-associated viral (rAAV) vectors are promising tools for gene therapy of genetic disorders, they remain mostly episomal and hence are lost during cell replication. For this reason, rAAV vectors capable of chromosomal integration would be desirable. Ribosomal DNA (rDNA) repeat sequences are overrepresented during random integration of rAAV. We therefore sought to enhance AAV integration frequency by including 28S rDNA homology arms into our vector design. A vector containing ~1 kb of homology on each side of a cDNA expression cassette for human fumarylacetoacetate hydrolase (FAH) was constructed. rAAV of serotypes 2 and 8 were injected into Fah-deficient mice, a model for human tyrosinemia type 1. Integrated FAH transgenes are positively selected in this model and rDNA-containing AAV vectors had a ~30× higher integration frequency than controls. Integration by homologous recombination (HR) into the 28S rDNA locus was seen in multiple tissues. Furthermore, rDNA-containing AAV vectors for human factor IX (hFIX) demonstrated increased transgene persistence after liver regeneration. We conclude that rDNA containing AAV vectors may be superior to conventional vector design for the treatment of genetic diseases, especially those associated with increased hepatocyte replication.

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Available from: Mark A Kay, Jun 28, 2014
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    • "Several studies have reported that efficient targeted gene addition at the rDNA 28s locus could be achieved based on viral transfer methods. However, because the majority of the integration was randomly located in the genome, the risks of the random integration were unavoidable for these methods [10–12]. Alternatively, based on a nonviral vector, in our previous studies, gene targeting at the rDNA 18S locus had been achieved, and the transgene could be stably expressed ectopically in targeted cells [13, 14]. "
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    ABSTRACT: Although recombinant adeno-associated virus (rAAV) vectors are proving to be efficacious in clinical trials, the episomal character of the delivered transgene restricts their effectiveness to use in quiescent tissues, and may not provide lifelong expression. In contrast, integrating vectors enhance the risk of insertional mutagenesis. In an attempt to overcome both of these limitations, we created new rAAV-rDNA vectors, with an expression cassette flanked by ribosomal DNA (rDNA) sequences capable of homologous recombination into genomic rDNA. We show that after in vivo delivery the rAAV-rDNA vectors integrated into the genomic rDNA locus 8-13 times more frequently than control vectors, providing an estimate that 23-39% of the integrations were specific to the rDNA locus. Moreover, a rAAV-rDNA vector containing a human factor IX (hFIX) expression cassette resulted in sustained therapeutic levels of serum hFIX even after repeated manipulations to induce liver regeneration. Because of the relative safety of integration in the rDNA locus, these vectors expand the usage of rAAV for therapeutics requiring long-term gene transfer into dividing cells.
    Full-text · Article · Sep 2012 · Molecular Therapy

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