AAV-mediated gene transfer in the perinatal period results in expression of FVII at levels that protect against fatal spontaneous hemorrhage

Abstract

We explored adeno-associated viral vector (AAV)-mediated gene transfer in the perinatal period in animal models of severe congenital factor VII (FVII) deficiency, a disease associated with early postnatal life-threatening hemorrhage. In young adult mice with plasma FVII < 1% of normal, a single tail vein administration of AAV (1 × 10(13) vector genomes [vg]/kg) resulted in expression of murine FVII at 266% ± 34% of normal for ≥ 67 days, which mediated protection against fatal hemorrhage and significantly improved survival. Codon optimization of human FVII (hFVIIcoop) improved AAV transgene expression by 37-fold compared with the wild-type hFVII cDNA. In adult macaques, a single peripheral vein injection of 2 × 10(11) vg/kg of the hFVIIcoop AAV vector resulted in therapeutic levels of hFVII expression that were equivalent in males (10.7% ± 3.1%) and females (12.3% ± 0.8%). In utero delivery of this vector in the third trimester to fetal monkeys conferred expression of hFVII at birth of 20.4% ± 3.7%, with a gradual decline to > 1% by 7 weeks. Re-administration of an alternative serotype at 12 months postnatal age increased hFVII levels to 165% ± 6.2% of normal, which remained at therapeutic levels for a further 28 weeks without toxicity. Thus, perinatal AAV-mediated gene transfer shows promise for disorders with onset of pathology early after birth.

Figure 1

FVII-deficient neonatal mice treated with an AAV8 vector-encoding murine FVII. (A) A schematic of the rAAV-LP1-mFVII construct consisting of the murine FVII cDNA under the control of a liver-specific LP1 promoter/enhancer element, followed by the SV40 virus large T Ag poly-A signal. (B) FVII activity as assessed by a chromogenic assay in plasma of low FVII mice after treatment with 2 × 10¹³ vg/kg AAV8-LP1-mFVII (broken line, N = 9) compared with control untreated low FVII animals (solid line, N = 3). Vertical arrow indicates virus administration on day 35. (C) Kaplan-Meier survival curve showing that low FVII mice treated with a single tail vein injection of AAV8-LP1-mFVII survived for at least 65 days after administration (100 days postnatal; broken line, N = 19), a significant (P < .0001) improvement over untreated mice (solid line, N = 23).

Figure 2

Construction and evaluation of human FVII AAV vectors. (A) Schematic of the human FVII constructs. In scAAV-LP1-FVII-wt, the coding region of the human FVII cDNA without the 3′ and 5′ UTRs was inserted into our scAAV backbone downstream of the LP1 and followed by the SV40 virus large T Ag poly-A signal. scAAV-LP1-FVII-coop is identical to scAAV-LP1-FVII-wt except that the wild-type human FVII cDNA has been replaced with a codon-optimized cognate. (B) Genomic configuration of the scAAV-LP1-FVII-wt and scAAV-LP1-FVII-coop provirus is shown under native (left) or alkaline (right) conditions.

Figure 3

Evaluation of AAV-mediated FVII expression. (A) FVII Ag (left side, ■) and activity (right side, ▩) in supernatant harvested from HepG2 cells 72 hours after the final transduction with scAAV8-LP1-hFVII-coop at an moi of 1 × 10⁵ vg/cell/d over 4 days. (B) Human FVII levels in plasma of male C57Bl/6 mice following a single tail vein administration of 2.8 × 10¹² vg/kg scAAV-encoding human FVII (circles = hFVII-coop, squares = hFVII-wt). (C) Top panels: Northern blot showing total human FVII mRNA levels in the liver of mice transduced with 2.8 × 10¹² vg/kg scAAV-LP1-FVII-wt (left) and scAAV-LP1-FVII-coop (right) compared with control untransduced animals (naive). Bottom image: Ethidium-stained agarose gel showing equal loading of liver RNA before transfer onto the nitrocellulose membrane. (D) Representative Western blot showing that human FVII-coop expressed following scAAV-mediated gene transfer (transduced mouse) has the same molecular weight as recombinant FVII and FVII detected in human pooled plasma. Serial dilution of recombinant FVII in naive mouse plasma suggests that the amount of human FVII in the experimental animal is > 1 μg/mL. (E) Human FVII levels in mice at 6 weeks after a single tail vein injection of between 1 × 10⁹-1 × 10¹² vg/kg scAAV-LP1-FVII-coop. Results are shown as means ± SEM.

Figure 4

AAV-mediated expression of codon-optimized hFVII in monkeys. (A) Human FVII levels over time in male (N = 3, dotted line) and female (N = 3, solid line) adult monkeys following a single peripheral vein administration of 4 × 10¹¹ vg/kg scAAV5-LP1-hFVII-coop. (B) Southern blot of DNA extracted from male and female rhesus monkey liver at 4 weeks after administration of scAAV5-LP1-hFVII-coop following digestion with BsRDI (double cutter) and probed with a hFVII-coop probe. As shown in the schematic, the probe is designed to detect the 1377- and 942-bp fragments expected from digest of head-to-tail concatemers.

Figure 5

Expression of hFVII in young rhesus monkeys following fetal administration of vector supernatant and readministration postnatally. (A) Plasma human FVII levels in young rhesus monkeys following administration of 4 × 10¹¹ vg/kg scAAV5-LP1-hFVII-coop prenatally and scAAV8-LP1-hFVII-coop at 1 year postnatal age. (B) Anti-AAV5 IgG levels in plasma after fetal administration of AAV5 LP1 hFVII coop. (C) Anti-AAV8 IgG levels following administration of scAAV8-LP1-hFVII-coop (second administration of vector) in monkeys at 1 year postnatal age.