Adenovirus-associated virus vector-mediated gene transfer in hemophilia B

Abstract

Background: Hemophilia B, an X-linked disorder, is ideally suited for gene therapy. We investigated the use of a new gene therapy in patients with the disorder.

Methods: We infused a single dose of a serotype-8-pseudotyped, self-complementary adenovirus-associated virus (AAV) vector expressing a codon-optimized human factor IX (FIX) transgene (scAAV2/8-LP1-hFIXco) in a peripheral vein in six patients with severe hemophilia B (FIX activity, <1% of normal values). Study participants were enrolled sequentially in one of three cohorts (given a high, intermediate, or low dose of vector), with two participants in each group. Vector was administered without immunosuppressive therapy, and participants were followed for 6 to 16 months.

Results: AAV-mediated expression of FIX at 2 to 11% of normal levels was observed in all participants. Four of the six discontinued FIX prophylaxis and remained free of spontaneous hemorrhage; in the other two, the interval between prophylactic injections was increased. Of the two participants who received the high dose of vector, one had a transient, asymptomatic elevation of serum aminotransferase levels, which was associated with the detection of AAV8-capsid-specific T cells in the peripheral blood; the other had a slight increase in liver-enzyme levels, the cause of which was less clear. Each of these two participants received a short course of glucocorticoid therapy, which rapidly normalized aminotransferase levels and maintained FIX levels in the range of 3 to 11% of normal values.

Conclusions: Peripheral-vein infusion of scAAV2/8-LP1-hFIXco resulted in FIX transgene expression at levels sufficient to improve the bleeding phenotype, with few side effects. Although immune-mediated clearance of AAV-transduced hepatocytes remains a concern, this process may be controlled with a short course of glucocorticoids without loss of transgene expression. (Funded by the Medical Research Council and others; ClinicalTrials.gov number, NCT00979238.).

Figure 1. Factor IX (FIX) Activity after Peripheral-Vein Infusion of the Adenovirus-Associated Virus (AAV) Vector in the Six Study Participants

A one-stage clotting assay was used to determine FIX coagulation activity at prespecified time points (arrows) after the administration of the AAV vector (scAAV2/8-LP1-hFIXco). Panels A and B show FIX levels in Participants 1 and 2, respectively, who received the low dose of vector (2×10¹¹ vector genomes [vg] per kilogram of body weight). Panels C and D show FIX levels in Participants 3 and 4, respectively, who received the intermediate dose of vector (6×10¹¹ vg per kilogram). Panels E and F show both FIX levels and alanine aminotransferase (ALT) levels in Participants 5 and 6, respectively, who received the high dose of vector (2×10¹² vg per kilogram). The duration of treatment with prednisolone (Pred), which was initiated at a dose of 60 mg per day and then gradually tapered, is also shown.

Figure 2. Humoral Immune Response in Participant 1

The profile of the humoral immune response in Participant 1 is representative of the responses seen in the other participants. Plasma samples obtained after peripheral-vein infusion of the scAAV2/8-LP1-hFIXco vector were analyzed with the use of an enzyme-linked immunosorbent assay for the presence of AAV8-specific IgM antibodies, total IgG antibodies, and IgG isotypes. Data for the IgG1 isotype are shown. IgG2 and IgG4 levels did not increase above baseline (data not shown).

Figure 3. Cellular Immune Response in the Six Study Participants

Results of the interferon-γ enzyme-linked immunosorbent spot assay for capsid-specific T-cell responses are shown as the maximum number of spot-forming units (SFU) per 1 million peripheral-blood mononuclear cells (PBMCs) in response to AAV8-capsid peptide pools. The horizontal lines in each panel represent the threshold for positivity, defined as three times the T-cell response for the negative control (medium only) and as at least 50 SFU per 1 million PBMCs. Liver-enzyme levels were elevated in Participant 5 at weeks 8 and 9 and in Participant 6 at week 9. In Participant 6, poor cell recovery and viability were noted at weeks 4 through 8.