Coexpression of factor VIII heavy and light chain adeno-associated viral vectors produces biologically active protein

Abstract

We are interested in using recombinant adeno-associated viral vectors in the treatment of hemophilia A. Because of the size constraints of recombinant adeno-associated viral vectors, we delivered the heavy and light chains of the human factor 8 (hFVIII) cDNA independently by using two separate vectors. Recombinant AAV vectors were constructed that utilized the human elongation factor 1alpha promoter, a human growth factor polyadenylation signal, and the cDNA sequences encoding either the heavy or light chain of hFVIII. Portal vein injections of each vector alone, a combination of both vectors, or a hFIX control vector were performed in C57BL/6 mice. An ELISA specific for the light chain of hFVIII demonstrated very high levels (2-10 microgram/ml) of protein expression in animals injected with the light chain vector alone or with both vectors. We utilized a chromogenic assay in combination with an antibody specific to hFVIII to determine the amount of biologically active hFVIII in mouse plasma. In animals injected with both the heavy and light chain vectors, greater than physiological levels (200-400 ng/ml) of biologically active hFVIII were produced. This suggests that coexpression of the heavy and light chains of hFVIII may be a feasible approach for treatment of hemophilia A.

Figure 1

Map of rAAV-hFVIII-HC and rAAV-hFVIII-LC vectors. Upper line in each illustration represents the gene structure of the vectors, and the lower line in each represents the structure of the hFVIII protein domains encoded by the vectors. ITR, AAV inverted terminal repeat; EF1α Pro/Intron 1, human polypeptide elongation factor 1α gene promoter and first intron; hFVIII-HC, human FVIII cDNA (hFVIII base pairs 1–2292); hFVIII-LC, human FVIII cDNA (hFVIII base pairs 1–57 and 4744–7053); hGH PA, human growth hormone polyadenylation signal; SS, human FVIII signal sequence; A1, A2, “B”, A3, C1, C2, complete and incomplete (“‘) protein domains of the hFVIII protein.

Figure 2

Cotransduction of 293 cells with rAAV-hFVIII-HC and rAAV-hFVIII-LC. 293 cells were plated on culture slides and, 48 hr later, were transduced at a multiplicity of infection of 3 × 10⁴ particles per cell with rAAV-hFVIII-LC (A), rAAV-hFVIII-HC (B), or both vectors (C). Forty-eight hours posttransduction, the cells were fixed and stained with a fluorescently labeled anti-hFVIII light chain antibody (green; 1:250) and a fluorescently labeled anti-hFVIII heavy chain antibody (red; 1:1,000) and counterstained with 4′,6-diamidino-2-phenylindole. Colocalization of both the heavy and light chains of hFVIII is evident by the yellow staining.

Figure 3

Expression of human FVIII in mouse plasma. C57BL/6 mice were injected via the portal vein with either 3 × 10¹¹ particles of rAAV-hFVIII-HC, 3 × 10¹¹ particles of rAAV-hFVIII-LC, 3 × 10¹¹ particles of both rAAV-hFVIII-HC and rAAV-hFVIII-LC, or 3 × 10¹¹ particles of rAAV-hFIX. Blood samples were collected via the retroorbital, and plasma was analyzed by ELISA for the level of hFVIII light chain antigen.

Figure 4

Southern blot analysis of liver DNA. Liver DNA (20 μg) extracted 8 weeks postinjection from mice injected via the portal vein with 3 × 10¹¹ particles of rAAV-hFVIII-LC (lane 1), rAAV-hFIX (lane 2), rAAV-hFVIII-HC (lane 3), or both rAAV-hFVIII-HC and rAAV-hFVIII-LC (lane 4) was digested with BglII and hybridized with a probe specific for the light chain of hFVIII (A) or the heavy chain of hFVIII (B). Copy number controls were generated by spiking BglII-digested naïve mouse liver DNA with the corresponding plasmids at ratios of 10, 5, 1, 0.1, and 0.01 copies per diploid genome (lanes 5–9).

Figure 5

Northern blot analysis of liver RNA. Liver RNA (10 μg) extracted 8 weeks postinjection from uninjected mice (lane 1) or mice injected via the portal vein with 3 × 10¹¹ particles of rAAV-hFVIII-LC (lane 3), rAAV-hFVIII-HC (lane 4), both rAAV-hFVIII-HC and rAAV-hFVIII-LC (lane 2), or rAAV-hFIX (lane 5) was hybridized with a probe specific for the light chain of hFVIII (A) or the heavy chain of hFVIII (B).