Long-term correction of inhibitor-prone hemophilia B dogs treated with liver-directed AAV2-mediated factor IX gene therapy

Abstract

Preclinical studies and initial clinical trials have documented the feasibility of adenoassociated virus (AAV)-mediated gene therapy for hemophilia B. In an 8-year study, inhibitor-prone hemophilia B dogs (n = 2) treated with liver-directed AAV2 factor IX (FIX) gene therapy did not have a single bleed requiring FIX replacement, whereas dogs undergoing muscle-directed gene therapy (n = 3) had a bleed frequency similar to untreated FIX-deficient dogs. Coagulation tests (whole blood clotting time [WBCT], activated clotting time [ACT], and activated partial thromboplastin time [aPTT]) have remained at the upper limits of the normal ranges in the 2 dogs that received liver-directed gene therapy. The FIX activity has remained stable between 4% and 10% in both liver-treated dogs, but is undetectable in the dogs undergoing muscle-directed gene transfer. Integration site analysis by linear amplification-mediated polymerase chain reaction (LAM-PCR) suggested the vector sequences have persisted predominantly in extrachromosomal form. Complete blood count (CBC), serum chemistries, bile acid profile, hepatic magnetic resonance imaging (MRI) and computed tomography (CT) scans, and liver biopsy were normal with no evidence for tumor formation. AAV-mediated liver-directed gene therapy corrected the hemophilia phenotype without toxicity or inhibitor development in the inhibitor-prone null mutation dogs for more than 8 years.

Figure 1

Coagulation parameters after liver-directed AAV2-mediated FIX gene therapy in hemophilia B dogs. The ACT (A), WBCT (B), aPTT (C), FIX activity levels (D), and FIX antigen concentration (E) as a function of time after AAV2/cFIX vector administration in hemophilia B dogs, Brad (●) and Semillon (). Normal ranges of 1 to 2 minutes for ACT, 6 to 10 minutes for WBCT, and 8.0 to 14.4 seconds for aPTT are indicated by the shaded bars.

Figure 2

Coagulation parameters after muscle-directed AAV2-mediated FIX gene therapy in hemophilia B dogs. The ACT (A), WBCT (B), and aPTT (C) as a function of time after vector administration in hemophilia B dogs, Wilbur (♦) and Sauvignon (). The arrows indicate cyclophosphamide administration to Wilbur before and 2, 4, and 6 weeks after vector administration. The WBCT test was terminated at 60 minutes if a clot had not formed for the first 15 weeks. After 15 weeks, the WBCT test was terminated after 20 minutes if a clot had not formed.

Figure 3

Vector persistence in hemophilia B dogs after liver-directed gene therapy. Hepatic biopsies, DNA isolation, FIX PCR, and microcapillary electrophoresis were as described. The fragment at 174 bp is the endogenous mutant FIX amplicon, and the fragment at 179 bp is the AAV vector-derived normal canine FIX amplicon. The relative percentage of each fragment is indicated (%) to the right of each peak. Amplification of DNA from peripheral blood and sperm DNA generated only the 174-bp fragment (not shown).

Figure 4

Accumulation of vector-derived normal FIX mRNA in hemophilia B dogs, Brad and Semillon, after liver-directed gene therapy. Hepatic biopsies, RNA isolation, cDNA synthesis, PCR, and microcapillary electrophoresis were as described.

Figure 5

LAM-PCR analysis of liver biopsy DNA from Brad and Semillon. Note absence of unique bands in samples from Brad and Semillon.

Figure 6

Bethesda assay/lymphocyte proliferation after FIX challenge. Hemophilia B dogs receiving muscle- or liver-directed AAV-mediated gene therapy were challenged with 2 infusions of cFIX. After challenge, Bethesda assays (A) and lymphocyte proliferation assays (B) were performed.

Figure 7

CT and histopathologic evaluation of hemophilia B dogs after liver-directed AAV gene therapy. After contrast sagittal CT sections of hemophilia B dog Brad (A) and hemophilia B dog Semillon (B) livers. H&E sections (×200) of liver biopsy obtained 6 years after vector administration in Brad (C) and Semillon (D). The CT evaluation, liver biopsy, and histologic processing were as described. GB indicates gall bladder.