AAV-1–mediated gene transfer to skeletal muscle in humans results in dose-dependent activation of capsid-specific T cells by Federico Mingozzi, Janneke J. Meulenberg, Daniel J. Hui, Etiena Basner-Tschakarjan, Nicole C. Hasbrouck, Shyrie A. Edmonson, Natalie A. Hutnick, Michael R. Betts, John J. Kastelein, Erik S. Stroes, and Katherine A. High Blood Volume 114(10):2077-2086 September 3, 2009 ©2009 by American Society of Hematology

Plasma TG and serum CPK levels in subject E Plasma TG and serum CPK levels in subject E. Vertical dotted line represents time of vector administration; TG levels are expressed in millimoles per liter (mM) and CPK in international units per liter (IU/L). *Positive capsid IFN-γ ELISpot. Plasma TG and serum CPK levels in subject E. Vertical dotted line represents time of vector administration; TG levels are expressed in millimoles per liter (mM) and CPK in international units per liter (IU/L). *Positive capsid IFN-γ ELISpot. CPK: ULN 190 IU/L; TG: normal levels less than 2 mM, LPL study inclusion criteria more than 10 mM. Mean (± SD) TG prior to gene transfer (week −42 to 0) for subject E was 42.7 mM (± 9.3 mM) (shaded area). Federico Mingozzi et al. Blood 2009;114:2077-2086 ©2009 by American Society of Hematology

Capsid-specific IFN-γ ELISpot on PBMCs from subject E Capsid-specific IFN-γ ELISpot on PBMCs from subject E. (A) Capsid-specific IFN-γ ELISpot; results are expressed in SFU/106 PBMCs (average ± SD). Capsid-specific IFN-γ ELISpot on PBMCs from subject E. (A) Capsid-specific IFN-γ ELISpot; results are expressed in SFU/106 PBMCs (average ± SD). Error bars represent SD. P 1 to P 24, AAV peptide matrix pools. Positive pools are indicated (positive defined as at least 3-fold above the medium control and at least 50 SFU/106 PBMCs). Horizontal lines represent the cutoff for positivity. PMA indicates positive control (> 1000 SFU/106 PBMCs for all time points); M, medium only. (B) Matrix analysis of results. Peptides identified by positive pools at any time point are indicated in black boxes. Note that peptides 145 and 146 were included in the matrix pools 12 and 13. Federico Mingozzi et al. Blood 2009;114:2077-2086 ©2009 by American Society of Hematology

Functional characterization of capsid-specific T cells. Functional characterization of capsid-specific T cells. (A) Intracellular IFN-γ staining on subject E's week-6 PBMCs; numbers indicate the percentage of CD4−CD8+ T cells that are IFN-γ+. Cells are gated on forward and side scatter, on singlets, and on CD4−CD8+ T cells. (B) Representation of 3 independent intracellular IFN-γ staining experiments (average ± SD of % CD4−CD8+IFN-γ+ T cells). (C) Perforin ELISpot on subject E's PBMCs. Medium indicates medium only control; irrelevant, human retinal pigment epithelium 65 protein; AAV-1, AAV-1 capsids; and positive, CEF peptide pool. Data are expressed in SFU per 106 PBMCs. SFU per 106 PBMCs were compared between AAV-1 and medium control (P = .008), AAV-1, and irrelevant control (P = .007), and AAV-1 and positive controls (P > .05, not significant). Federico Mingozzi et al. Blood 2009;114:2077-2086 ©2009 by American Society of Hematology

Time course of T-cell responses to AAV-1 capsid measured by IFN-γ ELISpot. Time course of T-cell responses to AAV-1 capsid measured by IFN-γ ELISpot. Each horizontal bar represents an individual subject; time after intramuscular administration (in weeks) is indicated by the vertical lines. Gray bars indicate a negative ELISpot result; red bars indicate a positive ELISpot result. A time point was considered positive when the average SFU/106 PBMCs was higher than 3 × the medium control and at least 50 SFU/106 PBMCs for both the AAV-1 peptide pools and the AAV-1 empty particles antigens. The vector dose, in genome copies per kilogram (gc/kg), received by the subjects in the 2 cohorts is indicated on the left of the graph. *Negative for AAV-1 empty capsids. Federico Mingozzi et al. Blood 2009;114:2077-2086 ©2009 by American Society of Hematology

CD4+ and CD8+ T-cell depletion experiments. CD4+ and CD8+ T-cell depletion experiments. IFN-γ ELISpot on PBMCs, or CD4- or CD8-depleted fraction of PBMCs (CD4−CD8+ or CD4+CD8−, respectively). Samples were collected at week 19 (subject A) and week 12 (subject D). Results are expressed in SFU/106 cells as average (± SD) of 3 replicates. Peptide 79/80 indicates 15-mers previously identified in subject A by screening ELISpot; peptide 49, 15-mer previously identified in subject D by screening ELISpot; medium, negative control; and PMA, positive control (PMA and ionomycin). Federico Mingozzi et al. Blood 2009;114:2077-2086 ©2009 by American Society of Hematology

T-cell response to AAV capsid proteins. T-cell response to AAV capsid proteins. PBMCs were stimulated for 6 hours with a set of 15-mer peptides covering the AAV capsid protein sequence and cytokine responses assessed by polychromatic flow cytometry. (A) TNF-α response to capsid proteins in CD4+ and CD8+ T cells. Numbers in the top right corner indicate the percentage of responding CD4+ or CD8+ T cells. A minimum of 240 000 events were collected for each time point. (B) Immunophenotyping of responding TNF-α+ cells (red dots) compared with total CD4+ T cells (gray density plots). Cells were gated on forward and side scatter, CD3+ cells, CD14−CD16−CD20− cells. Cells were also stained with an Invitrogen amine reactive live/dead aqua dye. Federico Mingozzi et al. Blood 2009;114:2077-2086 ©2009 by American Society of Hematology

Polyfunctional analysis of T-cell responses to the AAV-1 capsid. Polyfunctional analysis of T-cell responses to the AAV-1 capsid. Concurrent expression of IL-2, IFN-γ, perforin, TNF-α, and CD107 is measured at baseline and after gene transfer. Pie charts represent the proportion of capsid-specific CD4+ (left) or CD8+ (right) T cells expressing 1, 2, 3, 4, or 5 markers. Cells were gated on forward and side scatter, CD3+ cells, CD14−CD16−CD20− cells. Cells were also stained with an Invitrogen amine reactive live/dead aqua dye. CD4+ or CD8+ T cells were analyzed for IL-2, IFN-γ, perforin, TNF-α, and CD107 expression in response to AAV-1 peptides against medium control. Federico Mingozzi et al. Blood 2009;114:2077-2086 ©2009 by American Society of Hematology

Anti–AAV-1 antibody subclass analysis. Anti–AAV-1 antibody subclass analysis. Serum levels of IgG1, IgG2, IgG3, IgG4, and IgM (average ± SD, ng/mL). ♦ indicates subjects with positive T-cell response to the AAV capsid measured by ELISpot (subjects A, D-E, and H); , subjects with no detectable T-cell response to the AAV capsid measured by ELISpot (subjects B-C, F-G); *P < .05. Federico Mingozzi et al. Blood 2009;114:2077-2086 ©2009 by American Society of Hematology