Macrophage Depletion via Clodronate Pretreatment Reduces Transgene Expression from AAV Vectors In Vivo

Figures

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  Figure 1. (A) Manual count of total numbers of splenocytes following treatment of mice with clodronate liposome treatment (CL) at two different volumes (n = 4 per group). There appeared to be a greater decline in total splenocyte numbers with the higher dose of clodronate liposomes, but this was not statistically significant. (B) Marginal zone macrophage count as assessed by flow cytometry following clodronate liposome treatment. Treatment with 33 μL of CL yielded a 65% reduction in numbers of marginal zone macrophages. This difference was statistically significant (p < 0.05). With a higher dose of 200 μL, marginal zone macrophage numbers could be reduced even further (80%).

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  Figure 2. Gross comparison of mice (n = 4) transduced systemically (via tail vein) with 1 × 1011 vg of AAV6 vector expressing human placental alkaline phosphatase (hPLAP) after being given an intravenous injection of clodronate liposomes or PBS, 48 h prior. Mice were euthanized 28 days post-AAV administration and tissues fixed and stained for hPLAP expression. There appeared to be a trend towards more expression in clodronate-untreated mice, particularly in the heart (A) and liver (B), but less so in the spleen (C). Images pictured are from individual mice. Yellow arrows point to regions of purple punctate staining representative of hPLAP expression.

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  Figure 3. Representative gross images of lung, lymph node, spleen, and kidney from clodronate-treated mice and control mice. Both groups (n = 4) were administered 1 × 1011 vg of AAV vector expressing human placental alkaline phosphatase (hPLAP) via tail vein injection and euthanized 28 days later. Tissues were fixed and stained for hPLAP expression. There did not appear to be grossly visible differences between these two groups of mice in these particular organs. Expression of hPLAP in these organs was relatively low.

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  Figure 4. Quantification of alkaline phosphatase activity following systemic administration of 1 × 1011 vg of an AAV6 vector expressing human placental alkaline phosphatase (hPLAP) with and without prior intravenous administration of clodronate. In the heart (p < 0.05) and liver (p < 0.005), a statistically significant difference could be observed in clodronate-treated and untreated mice, with untreated mice possessing on average ~4 fold more alkaline phosphatase activity. No significant difference could be observed in the levels of alkaline phosphatase activity in mouse lung and spleen.

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  Figure 5. Histochemical staining of sections from spleen and lymph node in clodronate-treated and control mice. (A) Representative tissue sections of spleen and lymph node from clodronate-treated mice and control mice. Both groups were administered 1 × 1011 vg of AAV vector expressing human placental alkaline phosphatase (hPLAP) via tail vein injection and euthanized 28 days later. Tissues were formalin fixed, paraffin embedded, and stained for hPLAP expression. Administration of clodronate appeared to allow improved AAV transduction of white pulp relative to control mice. hPLAP staining in the center of the white pulp could be observed for clodronate-treated mice, whereas with control mice, no such staining could be observed. The presence of marginal zone macrophages likely prevented efficient transduction of white pulp in control mice. Within the lymph node, staining seemed to be restricted to the periphery of the lymph node for control mice, whereas in clodronate-treated mice, staining could be observed in the center. (B) Quantification of hPLAP positive foci in the white pulp of spleens from clodronate-treated and control mice. Foci of hPLAP stained cells in the white pulp of the…

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  Figure 6. Representative tissue sections of the heart, liver, lung, and pancreas from clodronate-treated and control mice (PBS). Both groups were administered 1 × 1011 vg of AAV vector expressing human placental alkaline phosphatase (hPLAP) via tail vein injection and euthanized 28 days later. Tissues were formalin fixed, paraffin embedded, and stained for hPLAP expression. Images were taken at 10× magnification for all tissues except the pancreas, which was imaged at 4×. 4× scale bar = 200 μM, 10× scale bar = 100 μM.