683. In Vivo Expansion of Hepatocytes with Targeted rAAV Integration Results in a >100-Fold Increase of Transgene Expression
Authors: Sean Nygaard, Adi Barzel, Annelise Haft, Mark A. Kay, Markus Grompe
Journal: Molecular Therapy (2015)

The recent development of promoter-less transgene integration technology using 2A fusions promises enhanced safety over traditional gene therapies. These vectors target therapeutic transgenes to loci for highly expressed endogenous genes through homologous recombination. Due to the inherent low frequency of targeted integrations, transgene expression is limited and requires high vector doses. Here we describe a modified 2A fusion integration vector that facilitates in vivo selection and expansion of gene-targeted hepatocytes. In mice treated with this vector, factor IX transgene expression increased over 400-fold during selection. Selection was achieved by incorporating into the integration vector a genetic element that rescues gene-targeted hepatocytes from induced hepatotoxic conditions. Blocking Fah (fumarylacetoacetate hydrolase) leads to hepatotoxic accumulation of the enzyme's substrate fumarylacetoacetate (FAA). This toxicity can be rescued by inhibiting Hpd, an enzyme upstream of Fah. In Fah-deficient mice, hepatotoxity is readily manipulated by administering or withdrawing the drug NTBC which inhibits Hpd. Alternately, Hpd can be blocked with a shRNA. We've previously shown that in Fah-deficient mice, hepatocytes transduced with an Hpd-specific shRNA have a selective advantage when NTBC is withdrawn, leading to clonal expansion of transduced cells. This Hpd-specific shRNA was incorporated into the original albumin-targeting 2A integration vector as an shRNAmir. To prevent off-target expression, the shRNAmir was inserted without a promoter into an intron of the vector's 3' albumin homology arm. The resulting AAV-8 vector was administered to Fah-deficient neonates (P0, n=8) by facial vein injection at a dose of 4e11 Vg. At 4 weeks of age, plasma hF-IX levels measured 167 +/- 65 ng/ml. The mice were then cycled off NTBC to select for hepatocytes rescued through expression of the Hpd shRNAmir. Plasma hF-IX levels increased exponentially, reaching 46, 000 +/- 5, 000 ng/ml after 11 weeks of NTBC cycling (n=4). Three of the treated mice were cycled off NTBC for just 3 weeks, then maintained on NTBC for an additional 6 weeks. hF-IX levels remained steady at 400 – 1100 ng/ml, suggesting the integrated sequences were not toxic to hepatocytes. Control mice treated with the original albumin integration vector (lacking the HPD shRNAmir) showed no increase in plasma hF-IX levels during NTBC cycling. Administration of the selectable AAV to adult mice also resulted in increased hF-IX during NTBC withdrawal. Current efforts aim to replicate selection in wild-type mice using pharmacological inhibition of Fah. In summary, 2A fusion-based integration vectors promise significant safety advantages over traditional gene targeting methods. We've shown that incorporating selectable genetic elements into these vectors facilitates highly robust transgene expression which can be modulated in vivo. Selectable vectors could allow significantly lower vector doses, thereby enhancing the safety of such therapies even further.