Application of in- vitro-cultured primary hepatocytes to evaluate species translatability and AAV transduction mechanisms of action

Abstract

Recombinant adeno-associated virus (AAV) is an effective platform for therapeutic gene transfer; however, tissue-tropism differences between species are a challenge for successful translation of preclinical results to humans. We evaluated the use of in vitro primary hepatocyte cultures to predict in vivo liver-directed AAV expression in different species. We assessed whether in vitro AAV transduction assays in cultured primary hepatocytes from mice, nonhuman primates (NHPs), and humans could model in vivo liver-directed AAV expression of valoctocogene roxaparvovec (AAV5-hFVIII-SQ), an experimental gene therapy for hemophilia A with a hepatocyte-selective promoter. Relative levels of DNA and RNA in hepatocytes grown in vitro correlated with in vivo liver transduction across species. Expression in NHP hepatocytes more closely reflected expression in human hepatocytes than in mouse hepatocytes. We used this hepatocyte culture model to assess transduction efficacy of a novel liver-directed AAV capsid across species and identified which of 3 different canine factor VIII vectors produced the most transgene expression. Results were confirmed in vivo. Further, we determined mechanisms mediating inhibition of AAV5-hFVIII-SQ expression by concomitant isotretinoin using primary human hepatocytes. These studies support using in vitro primary hepatocyte models to predict species translatability of liver-directed AAV gene therapy and improve mechanistic understanding of drug-drug interactions.

Keywords: AAV transduction; AAV vectors; AAV5-hFVIII-SQ; hepatocytes; in vitro model; valoctocogene roxaparvovec.

Graphical abstract

Figure 1

Comparison of hFVIII-SQ vector DNA levels in transduced hepatocytes in vitro and in vivo In vitro data are mean ± SD, where each point represents an individual donor or a pool of donors. In vitro data were analyzed by two-way analysis of variance with a subsequent Student’s t test; within MOIs, there were no significant differences between species. In vivo data are mean ± SD. In vivo data were analyzed by two-way analysis of variance, and there were no significant differences between species except for mouse versus human. Cyno, cynomolgus monkey; FVIII, factor VIII; MOI, multiplicity of infection; SD, standard deviation. ∗p < 0.05.

Figure 2

Comparison of hFVIII-SQ vector RNA levels in transduced hepatocytes in vitro and in vivo. (A–C) Comparison of hFVIII-SQ RNA levels in transduced mouse hepatocytes in vitro and in vivo to hepatocytes of (A) cynomolgus monkeys, (B) rhesus monkeys, and (C) humans. ns, not significant: ∗p < 0.05, ∗∗p < 0.005, and ∗∗∗∗p < 0.0001. In vitro data are mean ± SD, where each point represents an individual donor or a pool of donors; data were analyzed by two-way analysis of variance with a subsequent Sidak’s multiple comparisons test. In vivo data are mean ± SD; data were analyzed by Student’s t-test. Cyno, cynomolgus monkey; FVIII, factor VIII; MOI, multiplicity of infection; SD, standard deviation.

Figure 3

Transduction of a novel AAV capsid in hepatocytes in vitro and in vivo (A and B) Comparison of transduction efficiency between a novel AAV capsid (AAV-BMN.L3-hAAT-bCG) with AAV5-hAAT-bCG using bCG as a reporter gene in mouse and cynomolgus monkey showing (A) DNA copy numbers and RNA transcripts in in vitro primary hepatocytes and (B) liver DNA copy numbers and RNA transcripts in vivo. Data are mean ± SD. AAV, adeno-associated virus; bCG, beta-chorionic gonadotropin; Cyno, cynomolgus monkey; SD, standard deviation.

Figure 4

Assessment of transduction of cFVIII constructs in hepatocytes in vitro and in vivo (A–E) Comparison of cFVIII DNA and RNA for 3 vector constructs for (A) in vitro mouse hepatocytes and (B) in vivo mouse liver, (C) canine FVIII protein in mouse plasma, (D) cFVIII DNA and RNA dose response for in vitro canine hepatocytes, and (E) FVIII activity in dog plasma. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗∗p < 0.0001. Data were analyzed with a one-way analysis of variance. Construct 1 is HLP-nco-cFVIII-SQ, construct 2 is HLP-co-cFVIII-SQ, and construct 3 is HLP-co-cFVIII-V3. Data are mean ± SD. In (E), data are from single dogs; one dog per construct. Co, codon-optimized; cFVIII, canine FVIII; FVIII, factor VIII; HLP, hybrid liver promoter; MOI, multiplicity of infection; nco, non–codon-optimized; SD, standard deviation.

Figure 5

Investigation of the effect of isotretinoin on FVIII expression using the in vitro model. (A) Clinical outcomes and isotretinoin use in a clinical trial participant who received a single 6 × 10¹³ vg/kg dose of valoctocogene roxaparvovec. (B–G) Effects of 2-day isotretinoin treatment on (B) FVIII DNA copies, (C) FVIII RNA transcripts, and (D) CYP26A1 gene expression, and 4 days post-isotretinoin removal on (E) FVIII DNA copies, (F) FVIII RNA transcripts, and (G) CYP26A1 gene expression in transduced human hepatocytes. ∗p < 0.05, ∗∗∗p < 0.005, and ∗∗∗∗p < 0.0001. Data are mean ± SD. Data were analyzed with a one-way ANOVA. FVIII activity was measured per CS assay (LLOQ, <3 IU/dL). In the 5- and 500-ng/mL groups, cells were collected for analysis after 2 days of treatment with isotretinoin. In the withdrawal groups, isotretinoin was applied for 2 days, then treatment was withdrawn for 4 days, and cells were collected for analysis. ALT, alanine aminotransferase; BL, baseline; CS, chromogenic substrate; FVIII, factor VIII; LLOQ, lower limit of quantitation; SD, standard deviation; Veh, vehicle.