Ribosomal DNA Integrating rAAV-rDNA Vectors Allow for Stable Transgene Expression

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.

Evaluating risks of insertional mutagenesis by DNA transposons in gene therapy

“…The first attempts to combine the targeted integration ability of zinc fingers with SB transposons consisted of adding a zinc-finger DNA-binding domain to SB transposase [198, 199], which resulted in diminished SB activity and marginal targeting. More recent approaches have employed either a targeted zinc-finger nuclease-mediated double-strand DNA break [200], the adeno-associated virus Rep protein [201] or targeting ribosomal RNA genes [202, 203]. Both strategies report better outcomes.…”

Therapeutic genome editing: prospects and challenges

“…AAV genomes containing transgenes flanked by homology to target loci are capable of stimulating HDR in the absence of a nuclease, albeit at lower rates 48,86,120-122 . Using this strategy one study targeted a factor IX cDNA to the highly expressed albumin locus, allowing for correction of the bleeding diathesis phenotype in factor IX deficient mice 62 .…”

Nonviral Gene Targeting at rDNA Locus of Human Mesenchymal Stem Cells

“…By using the nonviral delivery method, the absolute targeting frequency is more than 20-fold higher than that in HT1080 cells at HPRT , a most commonly targeted locus [34]. In previous reports, by including 28S rDNA homology arms into the vector design, the integration frequency of a recombinant adeno-associated viral vector in rat hepatocytes was enhanced by 30-fold [10]. The underlying mechanism appears to be the relatively high intrinsic activity of HR at the rDNA locus.…”

“…Targeting an exogenous gene into one or a few of the rDNA repeats may not cause loss of function effects of the rRNA genes owing to high copy number of this gene. These properties indicate that the rDNA locus may hold a high intrinsic homologous recombination (HR) activity and this locus is considered to be an ideal safe locus for transgene integration [10, 11]. Several studies have reported that efficient targeted gene addition at the rDNA 28s locus could be achieved based on viral transfer methods.…”

“…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].…”