Long-term expression of human coagulation factor VIII in a tolerant mouse model using the φC31 integrase system

Abstract

We generated a mouse model for hemophilia A that combines a homozygous knockout for murine factor VIII (FVIII) and a homozygous addition of a mutant human FVIII (hFVIII). The resulting mouse, having no detectable FVIII protein or activity and tolerant to hFVIII, is useful for evaluating FVIII gene-therapy protocols. This model was used to develop an effective gene-therapy strategy using the φC31 integrase to mediate permanent genomic integration of an hFVIII cDNA deleted for the B-domain. Various plasmids encoding φC31 integrase and hFVIII were delivered to the livers of these mice by using hydrodynamic tail-vein injection. Long-term expression of therapeutic levels of hFVIII was observed over a 6-month time course when an intron was included in the hFVIII expression cassette and wild-type φC31 integrase was used. A second dose of the hFVIII and integrase plasmids resulted in higher long-term hFVIII levels, indicating that incremental doses were beneficial and that a second dose of φC31 integrase was tolerated. We observed a significant decrease in the bleeding time after a tail-clip challenge in mice treated with plasmids expressing hFVIII and φC31 integrase. Genomic integration of the hFVIII expression plasmid was demonstrated by junction PCR at a known hotspot for integration in mouse liver. The φC31 integrase system provided a nonviral method to achieve long-term FVIII gene therapy in a relevant mouse model of hemophilia A.

FIG. 1.

Generation of huFVIII-R593C/E-16KO mice. (A) Breeding diagram shows how homozygous huFVIII-R593C/E-16KO mice were generated for use in the study. (B) PCR was conducted on tail DNA using primers that specifically amplify the hFVIII gene present in transgenic mice. Lanes 1–5, samples from experimental mice showing that mice 1 and 3–5 are positive for the hFVIII gene. MW, molecular weight ladder; +, positive control; −, negative control mouse DNA. (C) Southern blot of tail DNA digested with BamHI and hybridized to a 505-bp hFVIII probe. Lane 1, negative control (−/−); lanes 2–8, samples from experimental mice, showing that mice 2 and 6–8 were homozygous (+/+) and mice 3–5 were heterozygous (+/−).

FIG. 2.

φC31 integrase and hFVIII plasmids. pVI, the pVax backbone carrying the coding sequence for wild-type φC31 integrase under control of the cytomegalovirus promoter; pVhP2, a higher-efficiency φC31 integrase mutant; pVmI, pVax carrying an inactive mutant integrase; pVB8, liver-specific hFVIII plasmid; pVB8ii, liver-specific hFVIII plasmid containing an intron. The hFVIII plasmids contain the pVax backbone, φC31 integrase attB site, human α₁-antitrypsin (hAAT) promoter, B-domain–deleted hFVIII cDNA, with or without an intron, and the bovine growth hormone polyA sequence.

FIG. 3.

hFVIII activity in plasma of huFVIII-R593C/E-16KO mice injected with various plasmids. Mice were hydrodynamically injected with pVB8 alone, pVB8+pVmI, pVB8+pVI, pVB8+pVhP2, or a saline solution only. Plasma samples were assayed for hFVIII activity at the time point indicated. Expression of hFVIII was measured by activity assay. Values are means±SEM.

FIG. 4.

hFVIII activity in plasma of huFVIII-R593C/E-16KO mice injected with a single injection of pVB8+pVI, versus two injections of pVB8+pVI. Plasma samples were assayed for hFVIII activity at the time point indicated. Arrow indicates when the second injection was administered (day 30). Expression of hFVIII was measured by activity assay through the length of the study. Values are means±SEM. Asterisks denote values that differ statistically from a single dose of pVB8+pVI: **p<0.05 (Student's t test).

FIG. 5.

hFVIII activity, concentration, and bleeding times of huFVIII-R593C/E-16KO mice injected with various plasmids. Mice were hydrodynamically injected with pVB8ii alone, pVB8ii+pVmI, pVB8ii+pVI, pVB8+pVI, or a saline solution only. Plasma samples were assayed for hFVIII activity by activity assay and for expression by ELISA at the time points indicated. (A) Percentages are plotted of hFVIII activity from all groups throughout the experiment. Arrow denotes time point when a single carbon tetrachloride injection was given. Values are means±SEM. Asterisk denotes values that differ statistically from the pVB8ii only group: *p<0.05 (Student's t test). (B) hFVIII expression was assayed by ELISA at various time points. Arrow denotes time when a single carbon tetrachloride injection was given. Negative control animals were injected with a saline solution. Values are means±SEM. Asterisk denotes values that differ statistically from the pVB8ii only group: *p<0.05 (Student's t test). (C) Bleeding times following tail clip of huFVIII-R593C/E-16KO mice injected with various plasmids. Bleeding time was monitored over a 10-min time period. Asterisks denote values that differ statistically from pVB8ii+pVI: *p<0.05 or **p<0.005 (Student's t test).

FIG. 6.

Immune response to hFVIII. Immune response to hFVIII was measured by Bethesda assay on four huFVIII-R593C/E-16KO mice injected with 20 mg each of pVI+pVB8ii and an uninjected naive control. Mice were injected with plasmid DNA via hydrodynamic tail-vein injection, and plasma samples were collected at the times indicated. Asterisks denote values that differ from the naive group: **p<0.005 (Student's t test).

FIG. 7.

Genomic integration of pVB8ii into a genomic pseudo attP site in mouse liver. PCR demonstrates genomic integration of pVB8ii at a preferred pseudo attP site (mpsL1). Lanes 1–4, livers that were injected with pVB8ii+pVI; lane 5, liver that was injected with pVB8ii only; lane 6, liver that was injected with pVB8ii+pVmI. PCR band of 300 bp indicates plasmid integration at the mpsL1 pseudo att site. MW, DNA molecular weight marker;+, positive control from an animal known to have integration at mpsL1; Saline, liver injected with saline only.