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Clinical Trial
. 2011 Dec 22;365(25):2357-65.
doi: 10.1056/NEJMoa1108046. Epub 2011 Dec 10.

Adenovirus-associated Virus Vector-Mediated Gene Transfer in Hemophilia B

Free PMC article
Clinical Trial

Adenovirus-associated Virus Vector-Mediated Gene Transfer in Hemophilia B

Amit C Nathwani et al. N Engl J Med. .
Free PMC article


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; number, NCT00979238.).


Figure 1
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
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
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.

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