Sustained high expression of human FVII following AAV8-mediated gene delivery in mice

Abstract

Factor VII (FVII) deficiency is a rare bleeding disorder with a prevalence of approximately 1:300,000-500,000 individuals. We explored whether adeno-associated virus (AAV)-mediated gene therapy can achieve durable and functional expression of human FVII (hFVII) in vivo. Wild-type hFVII (hFVIIwt) and a naturally occurring splice variant designated hFVII(-22) (GenBank: NM_019616.4) were expressed under the control of the Apolipoprotein E-derived hepatic locus control region and the α1-anti-trypsin promoter. Expression cassettes were packaged in either recombinant AAV5 (rAAV5) or recombinant AAV8 (rAAV8). For both hFVIIwt and hFVII(-22), 5- to 20-fold higher plasma levels of hFVII could be obtained when rAAV8 was used as a vector as opposed to rAAV5. Interestingly, hFVII levels obtained by employing rAAV8 expressing the hFVII(-22) cDNA variant were approximately 10 times higher than those obtained using rAAV8 expressing hFVIIwt. Based on these results, we generated an rAAV8-based gene therapy vector encoding hFVII(-22) and evaluated long-term expression in vivo. Employing a vector dose of 0.8 × 10¹² genome copies (gc)/kg, we observed 48 weeks of functional hFVII expression which peaked at 16 IU/mL and stabilized at 7 IU/mL. These results support the pre-clinical development of AAV8-mediated delivery employing the splice-variant hFVII(-22) for patients suffering from FVII deficiency.

Keywords: AAV5; AAV8; FVII deficiency; augmentation therapy; gene therapy; liver-directed gene therapy; rare bleeding disorders.

Graphical abstract

Figure 1

In vivo biodistribution of AAV5, AAV8, and AAV9 (A) CAG-luciferase construct was packaged into rAAV5, rAAV8, or rAAV9 to test which wild-type serotype will target the liver more efficiently at a dose of 1.6 × 10¹² gc/kg in wild-type C57BL/6 mice (n = 5/group). (B) Representative individual pictures of the IVIS measurement taken at 1, 2, 4, 6, and 10 weeks post-injection. Animals were subjected to IVIS measurements after intraperitoneal injection of D-Luciferin. The AAV-mediated expressed transgene luciferase is detected by the emission of photons (scale bar is 1 × 10⁶ photons/cm²/s/sr within the ROI. (C) Quantified radiance has been transformed into logarithmic scale and presented as means (SD). Transformed data were analyzed by two-way ANOVA with Tuckey’s multiple comparisons test. ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Figure 2

Evaluation of AAV5- versus AAV8-mediated FVII expression (A) rAAV5 was packaged with either hFVIIwt or cohFVII(-22) under the control of the hAAT promoter and injected at a dose of 1.6 × 10¹³ gc/kg; while rAAV8 was packaged with the same constructs and injected at a dose of 1.6 × 10¹² gc/kg instead; (B) hFVIIwt levels in plasma of male C57BL/6 mice delivered by either rAAV5 or rAAV8; (C) human cohFVII(-22) levels in plasma of male C57BL/6 mice delivered by either rAAV5 or rAAV8. In both rAAV5- and rAAV8-mediated expression of hFVII recombinant protein, the hFVIIwt was outperformed by the shorter hFVII(-22) transcript. Data are presented as means (SD). See also Figure S2. Data were analyzed by Student’s t test. ∗∗∗∗p < 0.0001.

Figure 3

Biodistribution of AAV5 and AAV8 in mice tissues The chart summarized the relative distribution of both rAAV5 vectors and rAAV8 vectors copies per transduced organ detected by qPCR. For extended dataset, see also Figure S3.

Figure 4

Long-term expression using AAV8 capsid (A) AAV8 capsid was packaged with either FVIIwt and coFVII(-22) or two alternative codon-optimized versions, RBD-FVIIwt and RBD-FVII(-22), under the control of the hAAT promoter. Injection was performed at a dose of 0.8 × 10¹² gc/kg in wild-type C57BL/6 mice (n = 6/group). (B) Plasma levels of FVII after blood collection up to 48 weeks post-injection. No difference was observed between FVIIwt and the correspondent codon-optimized version RBD-FVIIwt as well as between coFVII(-22) and its codon-optimized version RBD-FVII(-22); however, both RBD-FVII(-22) and coFVII(-22) overperformed FVII(wt) and RBD-FVII(wt). For extended dataset, see also Figure S5. (C) FVII activity assay is used to test the same samples used for plasma antigen level. FVII activity assay is directly proportional to the amount of active protein present in the plasma of the injected mice. Dotted black line corresponds to mouse FVII threshold level (1 U/mL). Transcripts derived from FVII(-22) variants were confirmed to overperform FVIIwt variants. For extended dataset, see also Figure S6. Graphs show group means (SD); data were analyzed by two-way ANOVA with Tuckey’s multiple comparisons test. ns, non-significant; ∗∗∗∗p < 0.0001.

Figure 5

In vivo dosing study (A) To identify the optimal dosage, hAAT-RBD-FVII(-22) was packaged into AAV8. Injections were performed at a concentration of 0.3 × 10¹¹, 0.8 × 10¹¹, 1.6 × 10¹¹, or 0.8 × 10¹² gc/kg in wild-type C57BL/6 mice (n = 5–6/group); (B) FVII antigen level detected after injection at 0.3 × 10¹¹, 0.8 × 10¹¹, 1.6 × 10¹¹, or 0.8 × 10¹² gc/kg or 1.6 × 10¹¹, 0.8 × 10¹¹, and 0.3 × 10¹¹ gc/kg. Graphs show group means (SD); data were analyzed by two-way ANOVA with Tuckey’s multiple comparisons test. ns, non-significant; ∗∗∗∗p < 0.0001.

Figure 6

Anti-AAV8 antibodies in treated samples The bar graph showed plasma levels of anti-AAV8 antibodies in mice injected with rAAV8 capsids encoding for FVIIwt, coFVII(-22) RBD-FVIIwt, and RBD-FVII(-22), under the control of the hAAT promoter, after 6, 12, and 48 weeks post-injection as well as PBS-injected animals (n = 5–6/group). Graphs show group means (SD); data were analyzed by one-way ANOVA with Tuckey’s multiple comparisons test; ∗p < 0.05.