Illustration of a primate evolutionary tree. From this tree it is evident that chimpanzees are more closely related to modern humans than are any of the other primates. Chimpanzees and modern humans diverged from a common ancestor around 7 million years ago, this diagram also shows that chimpanzees are more closely related to humans than they are to any other primate including gorillas.

In vivo direct comparison AAV1 and AAV5 vectors in chimpanzee airways after aerosol delivery with a FL and RL dual reporter assay. The airways from four chimpanzees were aerosolized with a mixture of AAV1-CB-FL (1 × 10¹³ vg) and AAV5-CB-RL (1 × 10¹³ vg) with the use of a bronchoscopic microsprayer. Bronchial brushings were taken at (a) day 14, (b) day 45, and (c) day 90 to determine expression levels with a dual reporter luciferase assay. (c) Time course showing average luciferase activity for AAV1 and AAV5 expression in the four chimpanzees ± SEM. FL, firefly luciferase; AAV, adeno-associated virus; RL, Renilla luciferase; RLU, relative luminescence unit.

Humoral responses to AAV1 and AAV5 capsids after airway aerosol delivery. Total serum antibody levels to either AAV1 or AAV5 whole capsids were assayed by an indirect ELISA after coating plates with either AAV1 or AAV5 viral particles (a) group averages (b) individual chimpanzee values. AAV, adeno-associated virus; ELISA, enzyme-linked immunosorbent assay; IgG, immunoglobin G.

Validation of FL and RL dual reporter AAV1 and AAV5 vectors in primary human polarized airway epithelia. 5 × 10⁹ particles of each of the indicated rAAV vectors were applied alone or in combination to the apical membrane of polarized human airway epithelia and luciferase expression was assayed at 20 and 30 days post-infection. 1/50th of the crude cell lysate was used for luciferase activity measurement. Data represents the mean (±SEM) relative luciferase activity from six independent infections. (a) rAAV1 vectors encoding the FL (AAV1-CB-FL) or RL (AAV1-CB-RL) reporters were independently infected into polarized airway epithelia. These findings were used to calculate the differences in specific activity between the two luciferase transgenes using an average of both 20 and 30 day time points; RL has a 14-fold higher sensitivity than FL (1 FL unit = 14 RL units). (b) The comparison of rAAV1 and rAAV5 transduction with a single-RL reporter. Human airway epithelia were infected independently with either AAV1-CB-RL or AAV5-CB-RL vectors and transgene expression was assessed at the indicated time points. (c,d) The dual-luciferase reporter assay was applied to compare the transduction efficiency following coinfection of human airway epithelia with AAV1-CB-FL and AAV5-CB-RL. The raw data for relative light units from FL and RL measurement are shown in c and the corrected relative luciferase activities from the two different vectors are shown in d using a correction of 1 FL unit = 14 RL units as determined in a. Fold differences (~120-fold) in the efficiency of transduction for rAAV1 and rAAV5 vectors using the dual reporter assays (d) closely matched the data from the single reporter assays shown in b. FL, firefly luciferase; rAAV, recombinant adeno-associated virus; RL, Renilla luciferase.

Representative ex vivo IFN-γ ELISPOT assay results. Representative raw data is shown in terms of the numbers of SFCs per million PBMCs at each of the time points indicated. Each graph represents PBMCs from a single chimpanzee stimulated with either an AAV1 or AAV5 peptide pool as shown in the legend. PBMCs were subjected to an IFN-γ ELISPOT assay by stimulation with the respective AAV capsid pools or medium alone (no peptide). Each set of asterisks indicate responses that are statistically above background. Samples were compared using Student's t-tests, *P ≤ 0.05. (*Indicate responses that are over threefold of response from medium control). AAV, adeno-associated virus; IFN-γ ELISPOT, interferon-γ enzyme-linked immunosorbent spot; PBMC, peripheral blood mononuclear cell; SFC, spot forming cell.

Representative ex vivo IFN-γ ELISPOT assay results against transgene peptide library. Representative raw data is shown in terms of the numbers of SFCs per million PBMCs at day 110 post-rAAV delivery. PBMCs were subjected to an IFN-γ ELISPOT assay by stimulation with (a) the firefly luciferase or (b) Renilla luciferase peptide pool. Each set of asterisks indicate responses that are statistically above background (no peptide). Samples were compared using Student's t-tests, *P < 0.05. (*Indicate responses that are over threefold of response from medium control). AAV, adeno-associated virus; IFN-γ ELISPOT, interferon-γ enzyme-linked immunosorbent spot; PBMC, peripheral blood mononuclear cell; SFC, spot forming cell.