Sustained transgene expression despite T lymphocyte responses in a clinical trial of rAAV1-AAT gene therapy

Abstract

Alpha-1 antitrypsin (AAT) deficiency is well-suited as a target for human gene transfer. We performed a phase 1, open-label, dose-escalation clinical trial of a recombinant adeno-associated virus (rAAV) vector expressing normal (M) AAT packaged into serotype 1 AAV capsids delivered by i.m. injection. Nine AAT-deficient subjects were enrolled sequentially in cohorts of 3 each at doses of 6.9 x 10(12), 2.2 x 10(13), and 6.0 x 10(13) vector genome particles per patient. Four subjects receiving AAT protein augmentation discontinued therapy 28 or 56 days before vector administration. Vector administration was well tolerated, with only mild local reactions and 1 unrelated serious adverse event (bacterial epididymitis). There were no changes in hematology or clinical chemistry parameters. M-specific AAT was expressed above background in all subjects in cohorts 2 and 3 and was sustained at levels 0.1% of normal for at least 1 year in the highest dosage level cohort, despite development of neutralizing antibody and IFN-gamma enzyme-linked immunospot responses to AAV1 capsid at day 14 in all subjects. These findings suggest that immune responses to AAV capsid that develop after i.m. injection of a serotype 1 rAAV vector expressing AAT do not completely eliminate transduced cells in this context.

Conflict of interest statement

Conflict of interest statement: T.R.F. and B.J.B. hold patents related to this research, and any monies received have been donated to the Department of Pediatrics, College of Medicine at the University of Florida. T.R.F. and B.J.B. were also founders of Applied Genetic Technologies Corporation (AGTC), the study sponsor. They do not currently hold a position with the company, and any proceeds from this study are donated to the University of Florida. T.R.F.'s laboratory receives research funding from the Alpha 1 Foundation. M.L.B. receives research funding from, holds an endowed professorship from, and is a consultant for the Alpha 1 Foundation. The University of Florida owns an interest in the study agent and also owns stock in the sponsor of the study, AGTC. The Alpha 1 Foundation is closely affiliated with AlphaNet, which owns an interest in the study agent. J.M.W. is an inventor on patents including those involving AAV1 that have been licensed to various biopharmaceutical companies.

Figures

Fig. 1.

Time course of vector-mediated AAT expression and ELISPOT responses to AAV1 capsid peptides in (A) cohort 2 and (B) cohort 3. Serum M-specific AAT levels are plotted at the top of each panel and ELISPOT responses are plotted at the bottom of each panel. For subjects 201 and 303, who had discontinued AAT protein augmentation therapy 56 days before vector administration and resumed protein augmentation soon after day 90, AAT values are not plotted on day −1, day 3, or after day 90 but are described in the text. The day-365 sample for subject 202 could not be tested owing to severe hemolysis. ELISPOT responses are characterized as − (negative in both ex vivo and cultured assays), ± (positive in cultured assay but negative in ex vivo assay), or + (positive in both ex vivo and cultured assays). Samples for ELISPOT analysis were not available beyond day 90 for subjects 202, 203, and 302.

Fig. 2.

Time course of IFN-γ ELISPOT responses to pools of AAV1 capsid peptides or controls. PBMC were obtained at 1 or 2 times before (pre1 and pre2) and at 14, 30, 45, 60, 75, and 90 days (D14 through D90, respectively) and 425 or 321 days (Yr1) after vector administration and stimulated with each of 3 pools (A, B, and C) of AAV1 capsid peptides (15-mers overlapping by 10 aa) or with a positive control peptide pool (CEF). Responses that were significantly increased compared with medium alone (control) are indicated by an asterisk.

Fig. 3.

Evaluation of capsid-specific CD4+ and CD8+ T cell responses in subjects 301 and 302. (A) PBMC isolated at postinoculation day 45 were stimulated for 6 h with peptides derived from AAV-1 capsid (15-mers overlapping by 10 aa, bottom row) or were unstimulated (top row). Frequencies shown represent viable responding memory CD3+CD4+CD8- T cells within the respective quadrant gate after having gated out naïve cells (CCR7+CD45RO-). (B) Bar chart representing the functionality of AAV-specific CD4+ T cells. (C) PBMC isolated at postinoculation day 81 were stimulated for 6 h with peptides derived from AAV-1 capsid (15-mers overlapping by 10 aa, bottom row) or were unstimulated (top row). Frequencies shown represent viable responding memory CD3+CD8+CD4- T cells within the respective quadrant gate after having gated out naïve cells (CCR7+CD45RO-). (D) Bar chart representing the functionality of AAV-specific CD8+ T cells. Each bar represents the proportion of the total response positive or negative for the respective functions shown in the key below the x axis. Only bars with positive responses above the 0.005% threshold are shown.

Fig. 4.

Serum CK levels before and after vector administration.