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Gene-based FVIIa prophylaxis modulates the spontaneous bleeding phenotype of hemophilia A rats

Shannon M. Zintner, Juliana C. Small, Giulia Pavani, Lynn Dankner, Oscar A. Marcos-Contreras, Phyllis A. Gimotty, Mads Kjelgaard-Hansen, Bo Wiinberg and Paris Margaritis

Data supplements

Article Figures & Data

Figures

  • Figure 1.

    Construction and in vitro characterization of rat FVIIa. (A) To generate rat FVIIa, we introduced a short amino acid sequence (RKRRKR) at the expected site for rat FVII cleavage between the heavy and light chains (R152 and I153). The RKRRKR sequence is recognized and processed by the PACE/Furin intracellular protease, resulting in the secretion of 2-chain, activated rat FVII. A C-terminal HPC4 epitope tag was incorporated for immunoaffinity purification. (B) Polyacrylamide gel electrophoresis of recombinant rat FVIIa compared with rhFVIIa under reducing (R) and nonreducing (NR) conditions. The molecular size marker (M) is in kilodaltons. The heavy (∼35 kDa, H) and light (∼25 kDa, L) chains, as well as full-length (∼50 kDa, F), are indicated. (C) PT activity assay comparing activity of rat FVIIa (n = 7) relative to rhFVIIa (n = 7, 100%). Data are presented as mean ± standard deviation (SD). NS, nonsignificant difference (unpaired Student t test).

  • Figure 2.

    Efficacy of AAV-mediated gene transfer of rat FVIIa in hemostatically normal rats. (A) AAV8 vector used for infusion in hemostatically normal and HA rats. Inverted terminal repeats (ITRs) flank the expression cassette consisting of a human α1 antitrypsin (hAAT)/apolipoprotein E (ApoE) promoter/enhancer, a synthetic intron, the rat FVIIa cDNA, and a polyadenylation signal (pA). (B) Transgene expression (ng/mL) from AAV-rat FVIIa infusion (4.6 E13 [●, high dose] or 4.6 E11 vg/kg [▪, low dose]; n = 8 per dose) in hemostatically normal animals (baseline subtracted). (C) A PT clotting time assay before and after AAV infusion in hemostatically normal animals. Arrow indicates time of AAV administration. ****P < .0001 vs low dose 4.6 E11 vg/kg, using a repeated measures 2-way analysis of variance (ANOVA) with Bonferroni’s multiple comparison test. Data in panels B-C are presented as mean ± SD.

  • Figure 3.

    Bleeds in the HA rats. Each image represents a different animal experiencing a bleeding event. (A,F) Rear limb bleeds; (B,G) front limb bleeds (paw and shoulder, respectively); (C,H) lacerations or open wounds; (D,I) eye bleeds; and (E,J) “Other” including internal bleeding and urinary bleeds. Arrows indicate affected areas of the animals. BL, bladder; L, liver; S, spleen; ST, stomach. Animals in E-G and J were euthanized because of reaching ethical endpoints.

  • Figure 4.

    Efficacy of AAV-mediated gene transfer of rat FVIIa in HA rats. (A) Average transgene expression (ng/mL, baseline subtracted) after AAV infusion and (B) PT values before and after AAV infusion in HA animals at 4 weeks of age. HA-AAV ≥708 (▪, transgene expression in nanograms per milliliter; n = 15), HA-AAV <708 (●, transgene expression in nanograms per milliliter; n = 18) and untransduced control animals (WT [▼] controls [n = 12]; HA [▲] controls [n = 11]). Arrow indicates time of AAV administration. Thromboelastometry clot time (C) and α angle of recalcified whole blood (NATEM) (D). Each animal included in study had a minimum of 2 samples analyzed throughout the study. Sample sizes for panels C and D: WT controls (n = 9), HA controls (n = 8), HA animals with transgene expression ≥708 ng/mL (n = 12), and HA animals with transgene expression <708 ng/mL (n = 13). ****P < .0001; ***P < .001; **P < .01; *P < .05. Data are presented as mean ± SD. Experimental group comparison with HA control animals: repeated-measures 1-way ANOVA with Tukey’s multiple comparison test (A-B); 1-way ANOVA with Tukey’s multiple comparison test (C-D). NS, not significant.

  • Figure 5.

    Efficacy of AAV-mediated rat FVIIa transgene expression in the bleeding phenotype of HA rats. (A) Number of bleeding events exhibited by each animal over the course of the study. Each symbol represents an individual animal. (B) Average number of treatments received during each bleeding event in the HA, HA-AAV <708 (transgene expression in nanograms per milliliter), and HA-AAV ≥708 (transgene expression in nanograms per milliliter) cohorts. *P < .05; **P < .01, using a 1-way ANOVA with Tukey’s multiple comparison test after transformation of data. All data are presented as mean ± SD.

  • Figure 6.

    Tail vein transection to test for efficacy of expressed rat FVIIa. (A) Total volume of blood loss (milliliters) during a 45-minute period after tail vein injury. Each symbol represents an individual animal in each designated animal cohort. (B) The data in panel A are plotted as a function of transgene expression (ng/mL) vs total volume of blood loss (mL). Each symbol represents an individual animal. Gray horizontal bar indicates the range of blood loss values of WT control animals. Dashed lines indicate the 708 and 1250 ng/mL levels. ****P < .0001; **P < .01, using a 1-way ANOVA with Tukey’s multiple comparison test. All data are presented as mean ± SD.

  • Figure 7.

    Safety in HA animals overexpressing rat FVIIa. (A-E) Complete blood counts in HA, WT, HA-AAV <708 (transgene expression in nanograms per milliliter) and HA-AAV ≥708 (transgene expression in nanograms per milliliter) are shown: red blood cells (RBC), white blood cells (WBC), platelets (PLT), hemoglobin (Hb), and hematocrit (HCT) were monitored weekly throughout the study. (F) Fibrinogen (FIB) levels were monitored weekly in the same animal cohorts as in (A-E). The following symbols are used for each group in all panels: ▼, WT controls (n = 12); ▲, HA controls (n = 11); ●, HA – AAV ≥708 (transgene expression in nanograms per milliliter; n = 15); ▪, HA-AAV <708 (transgene expression in nanograms per milliliter; n = 18). Arrow indicates time of AAV administration. No statistical differences were observed among the experimental groups (repeated measures 1-way ANOVA with Tukey’s multiple comparison test). All data are presented as mean ± SD.

Tables

  • Table 1.

    Bleeding in HA controls and AAV-rat FVIIa-infused HA rats expressing <708 ng/ml (HA-AAV <708) or ≥708 ng/ml (HA-AAV ≥708) of rat FVIIa

    HA controls, n (%)HA-AAV <708, n (%)HA-AAV ≥708, n (%)*
    Animals with bleeds10 (91)15 (83)5 (33)
    Animals with no bleeds1 (9)3 (17)10 (67)
    Number of animals11 (100)18 (100)15 (100)
    • * P < .05 vs HA controls (Fisher's exact test).

    • P < .05 vs HA-AAV <708 (Fisher's exact test).

  • Table 2.

    The number of bleeds classified by type that were observed in the HA controls and AAV-rat FVIIa-infused HA rats expressing <708 ng/mL (HA-AAV <708) or ≥708 ng/mL (HA-AAV ≥708) of rat FVIIa

    Bleed typeHA controls, n (%)HA-AAV <708, n (%)HA-AAV ≥708, n (%)
    Rear limbs10 (50)10 (37)4 (44.5)
    Front limbs5 (25)13 (48)4 (44.5)
    Lacerations2 (10)1 (4)0 (0)
    Eyes2 (10)2 (7)0 (0)
    Other1 (5)1 (4)1 (11)
    Total bleeds20 (100)27 (100)9 (100)
    • There was no statistically significant difference in the location distribution among the 3 groups (Fisher's exact test, P = .933)