Figure 1.
The timing and experimental groups are schematically illustrated for each experiment in the study. The number of rats is indicated for each treatment group at the right of their treatment regimen schematic. (A) Experiment 1: Time course study of recombinant adeno-associated virus serotype 9-human erythropoietin (AAV9-hEPO) single administration. Rats were assigned into three groups and received injections of 2 µl of sterile PBS, AAV9-hEPO (5.9×1013 vg ml−1) or AAV9-Empty (5.9×1013 vg ml−1) into the right striatum. Rats in each treated group were further divided into three subgroups and sacrificed at three time points (4 days, 3 weeks and 6 months after intrastriatal injections). Rat brains were immunocytochemically processed for histological evaluation. (B) Experiment 2: Dose dependent study of AAV9-hEPO single administration. Rats were assigned into three groups and received injections of 2 µl of AAV9-hEPO at three different titers (5.9×1011 vg ml−1, 5.9×1012 vg ml−1, or 5.9×1013 vg ml−1) into the right striatum. After 3 weeks, rats were sacrificed and rat brains were immunocytochemically processed for histological evaluation. (C) Experiment 3: AAV9-hEPO re-administration study. Rats were assigned into four groups: Group 1 (denoted as M PBS-Str hEPO), Rats received the first injections of 200 µl of sterile PBS into the right quadriceps, and the second injections of 2 µl of AAV9-hEPO (5.9×1013 vg ml−1) into the right striatum 3 weeks later; Group 2 (M hEPO-Str hEPO), Rats received the first injections of 200 µl of AAV9-hEPO (5.9×1011 vg ml−1) into the right quadriceps, and the second injections of 2 µl of AAV9-hEPO (5.9×1013 vg ml−1) into the right striatum 3 weeks later; Group 3 (Str PBS-Str hEPO), Rats received the first injections of 2 µl of sterile PBS into the left striatum, and the second injections of 2 µl of AAV9-hEPO (5.9×1013 vg ml−1) into the right striatum 3 weeks later; and group 4 (Str hEPO-Str hEPO), Rats received the first injections of 2 µl AAV9-hEPO (5.9×1013 vg ml−1) into the left striatum, and the second injections of 2 µl of AAV9-hEPO (5.9×1013 vg ml−1) into the right striatum 3 weeks later. Peripheral blood samples were collected prior to sacrifice of rats for hematological, and antibody and immunoneutralization assays, and rat brains were immunocytochemically processed for histological evaluation 3 weeks after the second injections. Arrow heads indicate the time points of injections. M, muscular; Str, striatal.
Figure 2.
Temporal expression of transgene (human erythropoietin, hEPO) in the rat brain after intrastriatal injections of recombinant adeno-associated virus serotype 9-hEPO (AAV9-hEPO).
Photomicrographs were prepared from coronal sections through the striatum and processed for hEPO immunocytochemistry from representative rats 4 days (A and B), 3 weeks (C and D) and 6 months (E and F) after intrastriatal injections of 2 µl of AAV9-hEPO (5.9×1013 vg ml−1). B, D and F are high magnification photomicrographs for A, C and D, respectively. The scale bar = 1 mm in E also for A and C; 50 µm in F also for B and D. The arrows (D) indicate representative hEPO-immunoreactive (IR) cells. (G) Stereological quantification of hEPO-IR cells in the injected striatum of rats 4 days (open bar, n = 5), 3 weeks (gray bar, n = 5) and 6 months (black bar, n = 4) after injections. The data are presented as mean values±standard error of the mean (SEM). A one-factor analysis of variance (ANOVA) followed by Fisher's post hoc test was applied to compare hEPO-IR cell counts between different time points. * p<0.05 versus 4 days group.
Figure 3.
Dose dependent expression of transgene (human erythropoietin, hEPO) in the rat brain after intrastriatal injections of recombinant adeno-associated virus serotype 9-hEPO (AAV9-hEPO).
Photomicrographs were prepared from coronal sections through the striatum and processed for hEPO immunocytochemistry from representative rats received intrastriatal injections of 2 µl of AAV9-hEPO with a titer of 5.9×1011 vg ml−1 (A and B), 5.9×1012 vg ml−1 (C and D) or 5.9×1013 vg ml−1 (E and F) 3 weeks after injection. B, D and F are high magnification photomicrographs for A, C and E, respectively. The scale bar = 1 mm in E also for A and C; 50 µm in F also for B and D. The arrows (F) indicate representative hEPO-immunoreactive (IR) cells. (G) Stereological quantification of hEPO-IR cells in the injected striatum of rats received intrastriatal injections of 2 µl of AAV9-hEPO with a titer of 5.9×1011 vg ml−1 (open bar, n = 5), 5.9×1012 vg ml−1 (gray bar, n = 5) or 5.9×1013 vg ml−1 (black bar, n = 5) 3 weeks after injection. The data are presented as mean values±SEM. A one-factor analysis of variance (ANOVA) followed by Fisher's post hoc test was applied to compare hEPO-IR cell counts between different titer groups. * p<0.05 versus 5.9×1011 group. # p<0.05 versus the indicated groups.
Figure 4.
Transgene (human erythropoietin, hEPO) expression in the rat brain after intrastriatal injections of recombinant adeno-associated virus serotype 9-hEPO (AAV9-hEPO) from the M PBS-Str hEPO (A), M hEPO-Str hEPO (B), Str PBS-Str hEPO (C), and Str hEPO-Str hEPO (D and E) groups.
Rats first received intramuscular (200 µl, 5.9×1011 vector genomes, vg ml−1) or intrastriatal (2 µl, 5.9×1013 vector genomes, vg ml−1) injections for 3 weeks and then received AAV9-hEPO re-administration into the right striatum (2 µl, 5.9×1013 vector genomes, vg ml−1). Rats were sacrificed 3 weeks after AAV9-hEPO re-administration. Photomicrographs were prepared from coronal sections through the striatum and processed for hEPO immunocytochemistry. (E) is a high magnification photomicrograph of the right injected striatum for (D). The scale bar = 1 mm in D also for A, B and C; 50 µm for E. The arrows (E) indicate representative hEPO-immunoreactive (IR) cells. (F) Stereological quantification of hEPO-IR cells in the right injected striatum of rats from the M PBS-Str hEPO (open bar, n = 5), M hEPO-Str hEPO (gray bar, n = 5), Str PBS-Str hEPO (hatched bar, n = 5), and Str hEPO-Str hEPO (black bar, n = 5) groups 3 weeks after the second injections. The data are presented as mean values ± SEM. A one-factor analysis of variance (ANOVA) followed by Fisher's post hoc test was applied to compare hEPO-IR cell counts between different groups. * p<0.05 versus the M hEPO-Str hEPO group.
Figure 5.
Arbitrary rating scores of major histocompatibility complex (MHC) class I, MHC class II, cluster of differentiation 4 (CD4), CD8, complement receptors 3 (CR3) and glial fibrillary acidic protein (GFAP) immunostaining in the right striatum of rats received 2 µl of recombinant adeno-associated virus serotype 9-human erythropoietin (AAV9-hEPO, 5.9×1013 vg ml−1) (A), AAV9-Empy (5.9×1013 vg ml−1) (B), or sterile phosphate buffered saline (PBS) (C) at 4 days (open bars, n = 4), 3 weeks (gray bars, n = 5) and 6 months (black bars, n = 5) time points, and of rats received 2 µl of AAV9-hEPO at a titer of 5.9×1011 vg ml−1 (open bars, n = 5), 5.9×1012 vg ml−1 (gray bars, n = 5), or 5.9×1013 vg ml−1 (black bars, n = 5) 3 weeks after injections (D).
The bars represent the median values of rating scores, and the circles depict the individual values. A Kruskal-Wallis test followed by Mann-Whitney U tests was used to compare the rating scores between groups. * p<0.05 versus indicated groups.
Figure 6.
Accumulation of activated complement receptors 3 (CR3)-immunoreactive (IR) microglia and glial fibrillary acidic protein (GFAP)-IR astrocytes in the right injected striatum.
Photomicrographs were prepared from coronal sections through the striatum and processed for CR3 (A–D) and GFAP (F–I) immunocytochemistry in representative rats of the M PBS-Str hEPO (A and F), M hEPO-Str hEPO (B and G), Str PBS-Str hEPO (C and H) and Str hEPO-Str hEPO (D and I) groups 3 weeks after the second injections. The scale bar = 100 µm in I also for A–D and F–H. High magnification photomicrographs in the left bottom were prepared from the box areas in (D) and (I). (E) and (J) Arbitrary rating scores of CR3 (E) and GFAP (J) immunostaining in the right injected striatum of rats from the M PBS-Str hEPO, M hEPO-Str hEPO, Str PBS-Str hEPO and Str hEPO-Str hEPO groups. The bars represent the median values of rating scores, and the circles depict the individual values. Filled circles indicate the rats from which the photomicrographs were taken.
Figure 7.
Expression of major histocompatibility complex (MHC) class I and class II antigens in the right injected striatum.
Photomicrographs were prepared from coronal sections through the striatum and processed for MHC class I (A–D) and MHC class II (F–I) immunocytochemistry in representative rats of the M PBS-Str hEPO (A and F), M hEPO-Str hEPO (B and G), Str PBS-Str hEPO (C and H) and Str hEPO-Str hEPO (D and I) groups 3 weeks after the second injections. The scale bar = 100 µm in I also for A–D and F–H. High magnification photomicrographs in the left bottom were prepared from the box areas in (A) and (H). (E) and (J) Arbitrary rating scores of MHC class I (E) and MHC class II (J) immunostaining in the right injected striatum of rats from the M PBS-Str hEPO, M hEPO-Str hEPO, Str PBS-Str hEPO and Str hEPO-Str hEPO groups. The bars represent the median values of rating scores, and the circles depict the individual values. Filled circles indicate the rats from which the photomicrographs were taken. A Kruskal-Wallis test followed by Mann-Whitney U tests was used to compare the rating scores between groups. * p<0.05 versus indicated groups.
Figure 8.
Infiltration of cluster of differentiation 4 (CD4)-immunoreactive (IR) and CD8-IR T cells in the right injected striatum.
Photomicrographs were prepared from coronal sections through the striatum and processed for CD4 (A–D) and CD8 (F–I) immunocytochemistry in representative rats of the M PBS-Str hEPO (A and F), M hEPO-Str hEPO (B and G), Str PBS-Str hEPO (C and H) and Str hEPO-Str hEPO (D and I) groups 3 weeks after the second injections. The scale bar = 100 µm in I also for A–D and F–H. High magnification photomicrographs in the left bottom were prepared from the box areas in (C) and (I). (E) and (J) Arbitrary rating scores of CD4 (E) and CD8 (J) immunostaining in the right injected striatum of rats from the M PBS-Str hEPO, M hEPO-Str hEPO, Str PBS-Str hEPO and Str hEPO-Str hEPO groups. The bars represent the median values of rating scores, and the circles depict the individual values. Filled circles indicate the rats from which the photomicrographs were taken.
Table 1.
Summary of hEPO transduction and immune responses in the re-administration groups.
Figure 9.
The levels of anti-recombinant adeno-associated virus serotype 9 (AAV9) capsid (A) and anti-human erythropoietin (hEPO) (B) antibody in sera of rats received AAV9-hEPO re-administration were detected by enzyme-linked immunosorbent assay (ELISA) for the Control (age matched normal rats, open bar, n = 5), M PBS-Str hEPO (hatched bar, n = 5), M hEPO-Str hEPO (gray bar, n = 5), Str PBS-Str hEPO (black bar, n = 5) and Str hEPO-Str hEPO (striped bar, n = 5) groups.
The data are presented as mean values ± standard error of the mean (SEM). A one-factor analysis of variance (ANOVA) followed by Fisher's post hoc test was applied to make group comparisons. * p<0.05 versus the Control group; # p<0.05 versus the indicated groups. (C) Correlation between the total numbers(×105) of hEPO-IR cells in the right injected striatum of rats in the M PBS-Str hEPO, M hEPO-Str hEPO, Str PBS-Str hEPO and Str hEPO-Str hEPO groups, and the levels of anti-AAV9 capsid antibody in their sera. Total numbers of hEPO-IR cells were plotted against the values of optical density for anti-AAV9 capsid antibody from three independent experiments. Dots represent the individual values. Total numbers of hEPO-IR cells in the right injected striatum were found to be negatively correlated with the values of optical density for anti-AAV9 capsid antibody from three independent experiments (r = 0.50, p<0.05). M, muscular; PBS, phosphate buffered saline; Str, striatal.
Figure 10.
Immunoneutralization assay showing effects of sera from rats in the M hEPO-Str hEPO group on recombinant adeno-associated virus serotype 9 (AAV9)-or AAV8-mediated green fluorescent protein (GFP) transgene transduction in HEK293T cells.
The sera from five rats in the M hEPO-Str hEPO group in which rats received the first injections of AAV9-hEPO into the right quadriceps and the second injections of AAV9-hEPO into the right striatum 3 weeks later were collected 3 weeks after the second injections. The control sera from five age matched normal rats were also collected. 25 µl treated or control sera was added to 2 µl of virus (AAV9-GFP or AAV8-GFP, 1.0×1013 vg ml−1) and directly incubated on HEK293T cells for transduction. Photomicrographs were prepared using a fluorescent microscope from HEK293T cell cultures treated with AAV9-GFP +treatedsera (B), AAV9-GFP+control sera (E), AAV8-GFP+treated sera (H), or AAV8-GFP+control sera (J). (C) A phase contrast photomicrograph was prepared from the same field of (B). The scale bar = 100 µm in J also for B, C, E and H. HEK293T cells (1.0×106 ml−1) were also harvested from different treated cultures for flow cytometry analyses (A, D, G and I, representative flow cytometry charts). Note that treated sera blocked AAV9-mediated GFP transduction (A and B) but not AAV8-mediated GFP transduction on HEK293T cells (G and H). Incubation of the control sera had no effect on both AAV9- (D and E) and AAV8- (I and J) mediated GFP transduction. (F) Flow cytometry data from the AAV9-GFP + treated sera (open bar), AAV9-GFP + control sera (gray bar), AAV8-GFP + treated sera (hatched bar), and AAV8-GFP + control sera (black bar) treated cultures were summarized in the bar diagram. The data are presented as mean values ± standard error of the mean (SEM). A one-factor analysis of variance (ANOVA) followed by Fisher's post hoc test was applied to make group comparisons. * p<0.05 versus the indicated groups.
Figure 11.
Effect of recombinant adeno-associated virus serotype 9-human erythropoietin (AAV9-hEPO) re-administration on erythropoiesis.
Peripheral blood samples were collected 3 weeks after the second injections of AAV9-hEPO into the right striatum for hematological assays. The bar diagrams summarizes: (A) The number of red blood cells(×106 ml−1), (B) hemotocrit (%) of peripheral blood, and (C) hemoglobin (g/l) in peripheral blood per rat in the control (age matched normal rats, open bar, n = 5), M PBS-Str hEPO (striped bar, n = 5), M hEPO-Str hEPO (gray bar, n = 5), Str PBS-Str hEPO (hatched bar, n = 5) and Str hEPO-Str hEPO (black bar, n = 5) groups. The data are presented as mean values±standard error of the mean (SEM). A one-factor analysis of variance (ANOVA) followed by Fisher's post hoc test was applied to make group comparisons. *, # p<0.05 versus the indicated groups. M, muscular; PBS, phosphate buffered saline; Str, striatal.