Peripheral transvenular delivery of adeno-associated viral vectors to skeletal muscle as a novel therapy for hemophilia B

Abstract

Muscle represents an important tissue target for adeno-associated viral (AAV) vector-mediated gene transfer of the factor IX (FIX) gene in hemophilia B (HB) subjects with advanced liver disease. Previous studies of direct intramuscular administration of an AAV-FIX vector in humans showed limited efficacy. Here we adapted an intravascular delivery system of AAV vectors encoding the FIX transgene to skeletal muscle of HB dogs. The procedure, performed under transient immunosuppression (IS), resulted in widespread transduction of muscle and sustained, dose-dependent therapeutic levels of canine FIX transgene up to 10-fold higher than those obtained by intramuscular delivery. Correction of bleeding time correlated clinically with a dramatic reduction of spontaneous bleeding episodes. None of the dogs (n = 14) receiving the AAV vector under transient IS developed inhibitory antibodies to canine FIX; transient inhibitor was detected after vector delivery without IS. The use of AAV serotypes with high tropism for muscle and low susceptibility to anti-AAV2 antibodies allowed for efficient vector administration in naive dogs and in the presence of low- but not high-titer anti-AAV2 antibodies. Collectively, these results demonstrate the feasibility of this approach for treatment of HB and highlight the importance of IS to prevent immune responses to the FIX transgene product.

Figure 1

Time course of circulating canine FIX and coagulation activity in naive hemophilia B dogs after ATVRX vector delivery. (A) Canine FIX (cFIX) antigen levels measured by ELISA. (B) Activated partial thromboplastin time (aPTT); s indicates seconds. (C) Whole blood clotting time (WBCT); m indicates minutes. (D) Creatinine phosphokinase (CPK) levels. Each line represents an individual dog: red indicates dogs from the low-dose cohort (1 × 10¹² vg/kg); black, dogs from the mid-dose cohort (3 × 10¹² vg/kg). Shaded gray areas represent indicate range of values for hemostatically normal animals. x axis represents time in days.

Figure 2

Canine FIX staining and histology of muscle biopsies from dog H48 (3 × 10¹² vg/kg) 2 years after ATVRX delivery of an AAV2-cFIX vector. Left panel, sites of muscle biopsy sampling, AAV-injected target limb: (A) semimembranosus; (B) vastus lateralis; (C) semitendinosus. (A-C) cFIX immunostaining of muscle biopsies collected at the sites indicated. (D) cFIX immunostaining of muscle biopsies from the contralateral, noninjected limb. (E) Hematotoxylin and eosin staining of the muscle biopsy collected from the target leg. Images are taken at 100× magnification.

Figure 3

Transient nonneutralizing anti-cFIX antibodies develop at high doses of of AAV2-cFIX vector delivered via ATVRX. (A) Canine FIX transgene antigen levels measured by ELISA. (B) Anti-cFIX IgG1 and IgG2 subclass antibody titer measured by ELISA. (C-D) Blood coagulation activity measured by WBCT (m indicates minutes) and aPTT (s indicates seconds). (E) Creatinine phosphokinase (CPK) levels. Shaded gray areas indicate the range of values for hemostatically normal animals. Vertical arrows represent normal pooled plasma administrations. x axis represents time in days.

Figure 4

Delivery of AAV2-cFIX via ATVRX with no IS. (A) Canine FIX antigen levels measured by ELISA. (B-C) Coagulation parameters measured by WBCT (m indicates minutes) and aPTT (s indicates seconds). (D-E) Anti-cFIX IgG1 and IgG2 subclass antibody titer measured by ELISA in J03 (D) and J62 (E). *Peak neutralizing antibody titer (1.5 BU at day 35 after ATVRX). Shaded gray areas represent range of values for hemostatically normal animals. Vertical arrows represent pooled normal plasma administrations. x axis represents time in days.

Figure 5

Readministration of AAV vectors by ATVRX in HB dogs previously exposed to AAV-2 vectors. (A-B) Canine FIX levels measured by ELISA and coagulation activity (WBCT in minutes [m]) in dogs D32 (red) and B46 (black) upon readminstration of an AAV2-cFIX vector via ATVRX. (D-E) Canine FIX levels measured by ELISA and coagulation activity (WBCT in minutes [m]) in dogs E59 (red) and H27 (black) upon readminstration of an AAV6-cFIX vector via ATVRX. (C,F) Creatinine phosphokinase (CPK) levels. x axis represents time in days.

Figure 6

Time course of canine FIX expression and coagulation activity in naive hemophilia B dogs after ATRVX delivery of an AAV6-cFIX vector. (A) Canine FIX antigen levels measured by ELISA. (B) Whole blood clotting time (WBCT); m indicates minutes. (C) Activated partial thromboplastin time (aPTT); s indicates seconds. x axis represents time in days.