Regional intravascular delivery of AAV-2-F.IX to skeletal muscle achieves long-term correction of hemophilia B in a large animal model

Abstract

In earlier work, we showed that adeno-associated virus-mediated delivery of a Factor IX gene to skeletal muscle by direct intramuscular injection resulted in therapeutic levels of circulating Factor IX in mice. However, achievement of target doses in humans proved impractical because of the large number of injections required. We used a novel intravascular delivery technique to achieve successful transduction of extensive areas of skeletal muscle in a large animal with hemophilia. We provide here the first report of long-term (> 3 years, with observation ongoing), robust Factor IX expression (circulating levels of 4%-14%) by muscle-directed gene transfer in a large animal, resulting in essentially complete correction of the bleeding disorder in hemophilic dogs. The results of this translational study establish an experimental basis for clinical studies of this delivery method in humans with hemophilia B. These findings also have immediate relevance for gene transfer in patients with muscular dystrophy.

Figure 1.

Histology of normal dog muscle 8 weeks after direct intramuscular injection of AAV-CMV-canine F.IX. (A) Anatomy of dog hindlimb, showing sites of biopsy. (B) Immunofluorescence staining for canine F.IX at injection site. (C) Hematoxylin and eosin staining of same site, showing normal histology. Vacuoles within muscle fibers are due to freeze artifact. (D) Immunofluorescence staining of uninjected muscle in same limb. For all panels, original magnification ×100.

Figure 2.

Histology of normal dog muscle 8 weeks after intravascular vector delivery by isolated limb perfusion. (A) Anatomy of dog hindlimb showing sites of biopsy. (B-F) Immunofluorescence staining for canine F.IX at multiple sites as indicated, showing extensive positive staining. (G-H) Hematoxylin and eosin staining of same samples. For all panels, original magnification ×100.

Figure 3.

Canine F.IX expression in hemophilia B dogs following ILP delivery of AAV-2 vector. (A) Canine F.IX antigen levels and (B) activated partial thromboplastin times (aPTTs) in plasma samples of hemophilia B dogs as a function of time after delivery of AAV-CMV-cF.IX. Dog D99 () was injected with 3.7 × 10¹² vg/kg, dog F57 (×) with 1.7 × 10¹² vg/kg, and dog H08 (▪) with 3.0 × 10¹² vg/kg, by isolated limb perfusion, accompanied by transient immunosuppression. Dog E59 (▴) was injected by peripheral vein with 2.9 × 10¹² vg/kg. Dog E60 (○) was injected at 3.9 × 10¹² vg/kg by ILP, without immunosuppression. Dog B48 () was injected by direct intramuscular injection with 3.4 × 10¹² vg/kg. Arrow denotes infusion of canine plasma, resulting in transient spike in cF.IX and drop in aPTT.

Figure 4.

Coagulation assays in E60, a dog treated by ILP without immunosuppression. (A) Canine F.IX antigen levels, (B) activated partial thromboplastin times (aPTTs), and (C) Bethesda assay (titer of neutralizing antibody) as a function of time after vector injection. Dog E60 was injected at 3.9 × 10¹² vg/kg and did not receive immunosuppresion. Gray band in panel B denotes range of normal aPTT; line at 50 seconds denotes hemophilic values.

Figure 5.

Immunofluorescence staining of muscle sections of the tibialis anterior of hemophilia B dogs injected 2 (dog F57) or 3 (dog D99) years earlier with AAV-CMV-canine F.IX by isolated limb perfusion. (A, D). Fluorescence of rhodamine (red) showing presence of canine F.IX in muscle fibers of ILP injected dogs F57 (A) or D99 (D). (B, E) Fluorescence of fluorescein isothiocyanate (FITC; green) showing presence of heparan sulfate proteoglycan (HSPG) in fibers of tibialis anterior of dog F57 (B) or D99 (E). (C, F) Simultaneous excitation of both fluorescence tags. (G) Fluorescence of rhodamine showing absence of canine F.IX expression in muscle biopsy from dog E59 (infusion of vector by peripheral vein). (H) Hematoxylin and eosin staining of same samples of dog (F57). For all panels, original magnification ×100.