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. 2018 Jul 23;29(12):1364-1375.
doi: 10.1089/hum.2018.080. Online ahead of print.

Optimized Adeno-Associated Viral-Mediated Human Factor VIII Gene Therapy in Cynomolgus Macaques

Jenny A Greig  1 , Jayme M L Nordin  1 , John W White  1 , Qiang Wang  1 , Erin Bote  1 , Tamara Goode  1 , Roberto Calcedo  1 , Samuel Wadsworth  2 , Lili Wang  1 , James M Wilson  1
Affiliations

Optimized Adeno-Associated Viral-Mediated Human Factor VIII Gene Therapy in Cynomolgus Macaques

Jenny A Greig et al. Hum Gene Ther. .

Abstract

Hemophilia A is a common hereditary bleeding disorder that is characterized by a deficiency of human blood coagulation factor VIII (hFVIII). Previous studies with adeno-associated viral (AAV) vectors identified two liver-specific promoter and enhancer combinations (E03.TTR and E12.A1AT) that drove high level expression of a codon-optimized, B-domain-deleted hFVIII transgene in a mouse model of the disease. This study further evaluated these enhancer/promoter combinations in cynomolgus macaques using two different AAV capsids (AAVrh10 and AAVhu37). Each of the four vector combinations was administered intravenously at a dose of 1.2 × 1013 genome copy/kg into five macaques per group. Delivery of the hFVIII transgene via the AAVhu37 capsid resulted in a substantial increase in hFVIII expression compared to animals administered with AAVrh10 vectors. Two weeks after administration of E03.TTR packaged within the AAVhu37 capsid, average hFVIII expression was 20.2 ± 5.0% of normal, with one animal exhibiting peak expression of 37.1% of normal hFVIII levels. The majority of animals generated an anti-hFVIII antibody response by week 8-10 post vector delivery. However, two of the five macaques administered with AAVhu37.E03.TTR were free of a detectable antibody response for 30 weeks post vector administration. Overall, the study supports the continued development of AAV-based gene therapeutics for hemophilia A using the AAVhu37 capsid.

Keywords: adeno-associated virus; factor VIII; hemophilia A; monkey.

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Figures

Figure 1.
Figure 1.
Human blood coagulation factor VIII (hFVIII) expression and anti-hFVIII antibody generation in individual cynomolgus macaques following administration of AAVrh10.E03.TTR.hFVIIIco-SQ.PA75. Five male rhesus macaques were administered intravenously (i.v.) with 1.2 × 1013 genome copies (GC)/kg of AAVrh10.E03.TTR.hFVIIIco-SQ.PA75. Macaques were bled biweekly to evaluate hFVIII expression in plasma by enzyme-linked immunosorbent assay (ELISA). Values are expressed as mean ± standard error of the mean (SEM; solid line). Anti-hFVIII immunoglobulin G (IgG) titers were also evaluated biweekly in plasma by ELISA (1/dilution; dashed line). The shaded area indicates the application of the immunosuppression protocol as required.
Figure 2.
Figure 2.
hFVIII expression and anti-hFVIII antibody generation in individual cynomolgus macaques following administration of AAVhu37.E03.TTR.hFVIIIco-SQ.PA75. Five male rhesus macaques were administered i.v. with 1.2 × 1013 GC/kg of AAVhu37.E03.TTR.hFVIIIco-SQ.PA75. Macaques were bled biweekly to evaluate hFVIII expression in plasma by ELISA. Values are expressed as mean ± SEM (solid line). Anti-hFVIII IgG titers were also evaluated biweekly in plasma by ELISA (1/dilution; dashed line). The shaded area indicates the application of the immunosuppression protocol as required.
Figure 3.
Figure 3.
hFVIII expression and anti-hFVIII antibody generation in individual cynomolgus macaques following administration of AAVrh10.E12.A1AT.hFVIIIco-SQ.PA75. Five male rhesus macaques were administered i.v. with 1.2 × 1013 GC/kg of AAVrh10.E12.A1AT.hFVIIIco-SQ.PA75. Macaques were bled biweekly to evaluate hFVIII expression in plasma by ELISA. Values are expressed as mean ± SEM (solid line). Anti-hFVIII IgG titers were also evaluated biweekly in plasma by ELISA (1/dilution; dashed line). The shaded area indicates the application of the immunosuppression protocol as required.
Figure 4.
Figure 4.
hFVIII expression and anti-hFVIII antibody generation in individual cynomolgus macaques following administration of AAVhu37.E12.A1AT.hFVIIIco-SQ.PA75. Five male rhesus macaques were administered i.v. with 1.2 × 1013 GC/kg of AAVhu37.E12.A1AT.hFVIIIco-SQ.PA75. Macaques were bled biweekly to evaluate hFVIII expression in plasma by ELISA. Values are expressed as mean ± SEM (solid line). Anti-hFVIII IgG titers were also evaluated biweekly in plasma by ELISA (1/dilution; dashed line). The shaded area indicates the application of the immunosuppression protocol as required.
Figure 5.
Figure 5.
Comparison of peak hFVIII expression in cynomolgus macaques. Five male cynomolgus macaques were administered i.v. with 1.2 × 1013 GC/kg of (A) AAVrh10.E03.TTR.hFVIIIco-SQ.PA75, (B) AAVhu37.E03.TTR.hFVIIIco-SQ.PA75, (C) AAVrh10.E12.A1AT.hFVIIIco-SQ.PA75, or (D) AAVhu37.E12.A1AT.hFVIIIco-SQ.PA75. Macaques were bled biweekly post vector administration to evaluate peak hFVIII expression in plasma by ELISA. (E) A comparison of average peak hFVIII expression across the vector treatment groups is shown. (F) The time to generation of anti-hFVIII antibodies following administration of each of the vectors is shown. Values are expressed as mean ± SEM. ns, not significant. *p < 0.05; **p < 0.01.
Figure 6.
Figure 6.
Partial thromboplastin time (PTT) values in vector-administered cynomolgus macaques. Five male cynomolgus macaques were administered i.v. with 1.2 × 1013 GC/kg of (A) AAVrh10.E03.TTR.hFVIIIco-SQ.PA75, (B) AAVhu37.E03.TTR.hFVIIIco-SQ.PA75, (C) AAVrh10.E12.A1AT.hFVIIIco-SQ.PA75, or (D) AAVhu37.E12.A1AT.hFVIIIco-SQ.PA75. Macaques were bled biweekly to evaluate PTT levels in plasma determined by Antech Diagnostics.
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