Analysis of Transduction Efficiency, Tropism and Axonal Transport of AAV Serotypes 1, 2, 5, 6, 8 and 9 in the Mouse Brain methods
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Methods
A) Confocal images from horizontal brain sections at -3 mm along the dorso-ventral axis in reference to Bregma taken from a mouse that received a co-injection of rAAV5 driving expression of GFP (green) and fluorescently labeled retrograde tracer CTB (red) in the dentate gyrus. B) same as A), for Bregma -4.5 mm. C) same as A), for Bregma -6.0 mm. Double labeled cells in the ventral entorhinal cortex appear yellow. White squares on the left indicate parts of images shown in higher magnification on the right. Scale bars: for whole sections: 1 mm; for insets: 250 µm. D) Cell counts of CTB-only labeled cells (red), GFP-only labeled cells (green) and double positive cells (yellow) in horizontal sections of the ipsilateral entorhinal cortex along the dorso-ventral axis of three individual mice.
Methods
A) Schematics of the ssAAV and scAAV constructs driving expression of an H2B-GFP fusion protein under the control of the neuronal human Synapsin 1 promoter that have been packaged in AAV8 capsids. B) High-magnification confocal fluorescence image of a coronal striatal section containing scAAV-transduced neurons that was stained with nuclear stain DAPI (blue) showing localization of the H2B-GFP fusion protein (green) in the nucleus. Scale bar: 25 µm. C) Schematic of a coronal brain section in reference to Bregma with a red box indicating the areas shown in D. D) Confocal fluorescence images of coronal striatal sections stained with DAPI (blue) prepared at increasing time points following injection of ssAAV (top row) or scAAV (bottom row). Green GFP expression in neurons becomes visible in ssAAV at eight days, whereas scAAV leads to earlier expression already at three days after in...
Production and purification of rAAV vectors
All rAAV vectors described were produced in HEK 293 cells by using a helper virus free, two-plasmid based production method [ ] for rAAV1, 2, 5, 6 and 8 and a three-plasmid based production method for packaging of rAAV9 vectors based on a commercially available AAV helper free system (Agilent Technologies, catalog no. 240071). HEK 293 cells were transfected by using the calcium phosphate method. 72 hours post transfection cells were harvested and collected by centrifugation (2500 x g, 20 min, 4°C). Cell pellets were resuspended in resuspension buffer (50 mM Tris, 2 mM MgCl2, pH 8.5) and lysed by three consecutive freeze/thaw cycles. For removal of genomic DNA cell lysates were incubated with benzonase (50 U/mL) for 1 hour at 37°C. Subsequently rAAV particles were precipitated with CaCl 2 (25 mM) followed by PEG precipitation (8% PEG-8000, 500 mM NaCl). After resuspension of...
Histological preparation and staining
For the investigation of transduction efficiency ( ), mice were sacrificed 21 days after virus injection, the brains dissected and immersion fixed in 4% PFA overnight. Fixed brains were cut on a vibratome (Leica, VT-1000) to 60 µm thick coronal slices, stained 30 minutes with a 5 mg/l 4', 6-diamidino-2-phenylindole (DAPI) solution and mounted on cover slips. In the experiments analyzing retrograde transport (, ) the same procedure was used, however, brains were cut to 70 µm thick horizontal slices. In the experiments analyzing the cell tropism and microglia induction ( ), 21 days (and 48h after LPS injection, respectively) after virus injection mice were anaesthetized with KM (as above) and transcardially perfused with 20 ml PBS containing 10 U/ml Heparin (Sigma, H3393) and 20 ml 4% PFA. Brains were dissected and post-fixed in 4% PFA overnight. Fixed brains were cut on a vi...
Histological preparation and staining
For experiments comparing the expression time course of scAAV and ssAAV the same procedure as described above was used, but brains were cut to 70 µm thick coronal slices, and stained 30 minutes with a 1 mg/l DAPI solution.
Results
We aimed to quantitatively analyze various AAV serotypes with respect to their characteristics to mediate transgene expression in the mouse brain. Towards this end we packaged the same DNA fragment that was flanked by two ITRs from AAV2 and contained a CMV promoter driving expression of a GFP reporter gene with capsids of serotypes 1, 2, 5, 6, 8 and 9. Packaging was performed in HEK293 cells and virus preparations were purified by PEG precipitation and subsequent CsCl gradient centrifugation (see methods). Titers of genome copies were analyzed using qPCR and were diluted in PBS to 9.6 * 10 11 viral genomes per ml. We determined the optimal injection volume, ensuring focal expression of the transgene in the targeted brain region, and still providing sufficient dynamic range for quantification of reporter gene expression, by injecting different volumes of rAAV5 virus solution into one o...
Time-course of transgene expression mediated by single-stranded and self-complementing rAAV
To characterize the time-course of rAAV-mediated expression in the mouse brain following scAAV and ssAAV transduction, we cloned constructs containing AAV2 ITRs in which expression of an H2B-GFP fusion protein is driven from the neuron-specific promoter of the human synapsin 1 gene leading to fluorescent labeling of the nuclei of neurons ( ). One construct was designed in the conventional way. The self-complementing construct was designed by deleting the terminal resolution site (trs) within the right ITR as described in the methods section. We packaged the two constructs in AAV8 capsids and injected 80 nl containing 8.1 * 10 7 genome copies in the striata of mice ( ). Mice were sacrificed at 0.5, 1, 3, 8 and 21 days following injection and brain slices containing the striatum were counter-stained with DAPI. We acquired confocal images in the green and blue fluorescence channel with i...
Supporting Information
Expression of GFP following striatal injections of various amounts of rAAV5. We injected various volumes of rAAV5 (9.6*10 11 VG/ml) driving expression of GFP from a CMV promoter into the striatum and analyzed transduction efficacy after 21 days of incubation. A) Schematic of a coronal brain section in reference to Bregma with a red box indicating the areas shown in B. B) Confocal images of brain sections of the left hemisphere stained with DAPI (blue) showing expression of the reporter GFP (green). Injection of larger volumes leads to increased GFP expression. An injection volume of 80 nl does not lead to saturation of expression. Note, injections of large volumes cause massive transduction also outside of the striatum. Scale bars: 1 mm. C) Mean fluorescence index (see methods) calculated for a region of interest encompassing the striatum following transduction with different volumes....
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A) Confocal images from horizontal brain sections at -3 mm along the dorso-ventral axis in reference to Bregma taken from a mouse that received a co-injection of rAAV5 driving expression of GFP (green) and fluorescently labeled retrograde tracer CTB (red) in the dentate gyrus. B) same as A), for Bregma -4.5 mm. C) same as A), for Bregma -6.0 mm. Double labeled cells in the ventral entorhinal cortex appear yellow. White squares on the left indicate parts of images shown in higher magnification on the right. Scale bars: for whole sections: 1 mm; for insets: 250 µm. D) Cell counts of CTB-only labeled cells (red), GFP-only labeled cells (green) and double positive cells (yellow) in horizontal sections of the ipsilateral entorhinal cortex along the dorso-ventral axis of three individual mice.
A) Schematics of the ssAAV and scAAV constructs driving expression of an H2B-GFP fusion protein under the control of the neuronal human Synapsin 1 promoter that have been packaged in AAV8 capsids. B) High-magnification confocal fluorescence image of a coronal striatal section containing scAAV-transduced neurons that was stained with nuclear stain DAPI (blue) showing localization of the H2B-GFP fusion protein (green) in the nucleus. Scale bar: 25 µm. C) Schematic of a coronal brain section in reference to Bregma with a red box indicating the areas shown in D. D) Confocal fluorescence images of coronal striatal sections stained with DAPI (blue) prepared at increasing time points following injection of ssAAV (top row) or scAAV (bottom row). Green GFP expression in neurons becomes visible in ssAAV at eight days, whereas scAAV leads to earlier expression already at three days after injection. Scale bars: 250 µm. E) Cumulative distributions of mean fluorescence in the green channel of individual nuclei identified based on DAPI signal. Each line corresponds to the distribution of a brain section from one injected mouse (region of interest with a fixed size corresponding to ap...
All rAAV vectors described were produced in HEK 293 cells by using a helper virus free, two-plasmid based production method [ ] for rAAV1, 2, 5, 6 and 8 and a three-plasmid based production method for packaging of rAAV9 vectors based on a commercially available AAV helper free system (Agilent Technologies, catalog no. 240071). HEK 293 cells were transfected by using the calcium phosphate method. 72 hours post transfection cells were harvested and collected by centrifugation (2500 x g, 20 min, 4°C). Cell pellets were resuspended in resuspension buffer (50 mM Tris, 2 mM MgCl2, pH 8.5) and lysed by three consecutive freeze/thaw cycles. For removal of genomic DNA cell lysates were incubated with benzonase (50 U/mL) for 1 hour at 37°C. Subsequently rAAV particles were precipitated with CaCl 2 (25 mM) followed by PEG precipitation (8% PEG-8000, 500 mM NaCl). After resuspension of PEG precipitates in 50 mM HEPES, 150 mM NaCl, 25 mM EDTA, pH 7.4 overnight at 4°C, rAAV particles were further purified by CsCl density gradient centrifugation. Fractions from CsCl density gradients were analyzed by measuring the refractory index. Samples within a refractory index ranging from...
For the investigation of transduction efficiency ( ), mice were sacrificed 21 days after virus injection, the brains dissected and immersion fixed in 4% PFA overnight. Fixed brains were cut on a vibratome (Leica, VT-1000) to 60 µm thick coronal slices, stained 30 minutes with a 5 mg/l 4', 6-diamidino-2-phenylindole (DAPI) solution and mounted on cover slips. In the experiments analyzing retrograde transport (, ) the same procedure was used, however, brains were cut to 70 µm thick horizontal slices. In the experiments analyzing the cell tropism and microglia induction ( ), 21 days (and 48h after LPS injection, respectively) after virus injection mice were anaesthetized with KM (as above) and transcardially perfused with 20 ml PBS containing 10 U/ml Heparin (Sigma, H3393) and 20 ml 4% PFA. Brains were dissected and post-fixed in 4% PFA overnight. Fixed brains were cut on a vibratome to 50 µm coronal slices and incubated for 2h at RT in PBS containing 10% normal goat serum (Jackson Immuno research, 005-000-121) and 1% Triton-X 100 (Sigma-Aldrich, T8787). Subsequently, slices were washed three times for 10 min with PBS and incubated with primary antibody (GABA (Sigma...
For experiments comparing the expression time course of scAAV and ssAAV the same procedure as described above was used, but brains were cut to 70 µm thick coronal slices, and stained 30 minutes with a 1 mg/l DAPI solution.
We aimed to quantitatively analyze various AAV serotypes with respect to their characteristics to mediate transgene expression in the mouse brain. Towards this end we packaged the same DNA fragment that was flanked by two ITRs from AAV2 and contained a CMV promoter driving expression of a GFP reporter gene with capsids of serotypes 1, 2, 5, 6, 8 and 9. Packaging was performed in HEK293 cells and virus preparations were purified by PEG precipitation and subsequent CsCl gradient centrifugation (see methods). Titers of genome copies were analyzed using qPCR and were diluted in PBS to 9.6 * 10 11 viral genomes per ml. We determined the optimal injection volume, ensuring focal expression of the transgene in the targeted brain region, and still providing sufficient dynamic range for quantification of reporter gene expression, by injecting different volumes of rAAV5 virus solution into one of the brain areas to be tested in this study, the striatum. Analysis of reporter gene expression 21 days post-surgery revealed that pressure-injection of 80 nl virus solution through a finely tapered glass capillary leads to focal transduction of solely the targeted brain area without the surroundin...
To characterize the time-course of rAAV-mediated expression in the mouse brain following scAAV and ssAAV transduction, we cloned constructs containing AAV2 ITRs in which expression of an H2B-GFP fusion protein is driven from the neuron-specific promoter of the human synapsin 1 gene leading to fluorescent labeling of the nuclei of neurons ( ). One construct was designed in the conventional way. The self-complementing construct was designed by deleting the terminal resolution site (trs) within the right ITR as described in the methods section. We packaged the two constructs in AAV8 capsids and injected 80 nl containing 8.1 * 10 7 genome copies in the striata of mice ( ). Mice were sacrificed at 0.5, 1, 3, 8 and 21 days following injection and brain slices containing the striatum were counter-stained with DAPI. We acquired confocal images in the green and blue fluorescence channel with identical settings ( ). We analyzed two injections per virus construct and time point corresponding to 15028±341 (mean±SEM) cells each, in a region of interest containing the transduction focus itself and a significant proportion of surrounding non-transduced tissue. The pipette track was u...
Expression of GFP following striatal injections of various amounts of rAAV5. We injected various volumes of rAAV5 (9.6*10 11 VG/ml) driving expression of GFP from a CMV promoter into the striatum and analyzed transduction efficacy after 21 days of incubation. A) Schematic of a coronal brain section in reference to Bregma with a red box indicating the areas shown in B. B) Confocal images of brain sections of the left hemisphere stained with DAPI (blue) showing expression of the reporter GFP (green). Injection of larger volumes leads to increased GFP expression. An injection volume of 80 nl does not lead to saturation of expression. Note, injections of large volumes cause massive transduction also outside of the striatum. Scale bars: 1 mm. C) Mean fluorescence index (see methods) calculated for a region of interest encompassing the striatum following transduction with different volumes. The images for this series of experiments were acquired with different settings as those shown in Figure 1A to avoid saturation of the GFP signal in mice injected with large volumes, thus the fluorescence index is not directly comparable to Figure 1A. Four hemispheres were injected and analyzed per...
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