1. Volume 18, Issue 4, Pages 715-724 (April 2010)
Comparison of AAV Serotypes for Gene Delivery to Dorsal Root Ganglion Neurons 
Matthew RJ Mason, Erich ME Ehlert, Ruben Eggers, Chris W Pool, Stephan Hermening, Angelina Huseinovic, Eric Timmermans, Bas Blits, Joost Verhaagen 
Molecular Therapy 
Volume 18, Issue 4, Pages (April 2010)
DOI: /mt
Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
GFP expression in sections of dorsal root ganglia (DRG) injected with vectors based on AAV serotypes 1, 2, 3, 4, 5, 6, and 8, and a lentiviral vector 2 weeks after injection. Sections were processed for immunohistochemistry for GFP (green) and βIII-tubulin (red). Representative sections are shown. AAV5 has the highest transduction rate, followed by AAV1 and AAV6. Bar = 100 µm. AAV, adeno-associated virus; GFP, green fluorescent protein; LV, lentiviral vector.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
AAV6 also transduces satellite cells in the DRG. Two confocal microscopy images are shown of sensory neurons in the DRG that were not transduced but are encapsulated by satellite cells that express GFP. (a,b) Satellite cells are stained with S100. (c,d) These satellite cells are also GFP+. (e,f) The neurons are visible with βIII-tubulin staining. (g,h) Composite images. Bar = 20 µm. DRG, dorsal root ganglia; GFP, green fluorescent protein.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
Example of the algorithmic identification of dorsal root ganglia neurons in tubulin and GFP-stained sections. (a) GFP and tubulin staining in a small area of a section. (b) Neuronal nuclei identified by the algorithm. (c) The mask is applied to the green channel and fluorescent intensity measured inside the selected areas. GFP, green fluorescent protein.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
Quantification of transduction rates and expression levels. (a–c) Quantification of GFP-expressing neurons after injection of viral vectors based on AAV serotypes 1, 2, 3, 4, 5, 6, and 8, and a lentiviral (LV) vector, 2 weeks after injection. (a) Percentages of neurons expressing GFP. Points represent transduction rates from individual ganglia. (b) Average expression level per DRG, expressed as a multiple of background fluorescence. For each ganglion, the mean log expression level of GFP-expressing cells was calculated. (c) Relative mRNA expression levels determined by qPCR. Values are shown on a log-2 scale. (d–f) Time course of GFP expression after injection of AAV1, AAV5, and AAV6. The 2-week time point is included again for clarity. (d) Percentages of neurons expressing GFP. Circles represent transduction rates from individual ganglia. (e) Average expression level per DRG is calculated as in b. (f) mRNA expression levels are determined by qPCR as in c. *P < 0.05, **P < 0.01, ***P < (ANOVA and Dunnett's T3 post hoc test, a–c; two-way ANOVA and Dunnett's post hoc test, d–f). Error bars are SEM, n = 8 DRG. AAV, adeno-associated virus; ANOVA, analysis of variance; DRG, dorsal root ganglia; GFP, green fluorescent protein.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
Histology of time course of GFP expression after injection of AAV5. (a–f) Sections were stained with GFP (green) and βIII-tubulin (red). (a,c,e) DRG neurons expressing GFP at 1 week (a), 4 weeks (c), and 12 weeks (e) after viral injection. (b,d,f) Sciatic nerves containing GFP+ fibers again at 1 week (b), 4 weeks (d), and 12 weeks (f) after injection. At 1 week, almost no GFP+ axons are present, whereas at 4 and 12 weeks, many axons are visible. (g) Horizontal section of superficial lumbar spinal cord, 4 weeks after AAV5, was injected into the left L4–5 DRG, stained for GFP (green) and GFAP (red). Many GFP+ fibers are visible in the dorsal roots and can be traced over long distances within the spinal cord. Bar = 100 µm (a,c,e), 50 µm (b,d,f), and 250 µm (g). AAV, adeno-associated virus; DRG, dorsal root ganglia; GFP, green fluorescent protein.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

7. Figure 6
Different neuronal subtypes are transduced by AAV5. Sections were triple-labeled for (a,d) βIII-tubulin, (b,e) GFP, and either (c) CGRP or (f) IB4. (a–c) CGRP-labeled neurons are GFP+. Arrows indicate neurons positive for both GFP and CGRP. (d–f) IB4-labeled neurons are also GFP+. Arrows indicate neurons positive for GFP and IB4. Bar = 50 µm. AAV, adeno-associated virus; GFP, green fluorescent protein.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

8. Figure 7
Transduction efficiencies of dorsal root ganglia neuronal subtypes. Quantification of (a) transduction rates and (b) expression levels in CGRP+ and IB4+ neurons, relative to the whole neuronal population, 4 weeks after injection of AAV1 or AAV5. Transduction rates and expression levels of CGRP+ cells are not different to the overall population. However, both serotypes show reduced transduction rates and expression levels of IB4+ cells. **P < 0.01 compared to the whole population.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions