Digital Droplet PCR Method for the Quantification of AAV Transduction Efficiency in Murine Retina

All experimental protocols were accepted by the Sabanci University ethics committee and experiments were conducted in accordance with the statement of 'The Association for Research in Vision and Ophthalmology' for the use of animals in research

1. Small scale AAV production¹²

1. Culture HEK293T cells using 15 cm plates in complete 10 mL of DMEM/10% FBS until 70-80% confluency.
2. Prepare the transfection mixture with 20 µg of helper plasmid (pHGT1-Adeno1), 7 µg of capsid plasmid and 7 µg of AAV2-CBA-tdTomato-WPRE vector and 136 µL of PEI solution (1 mg/mL) in 5 mL of DMEM.
3. Add the 5 mL prepared transfection mixture into to a cell culture dish containing 10 mL of culture media and incubate the transfected cells for 48-60 h at 37 °C.
4. Collect the media at 48-60 h post-transfection and digest it with DNase I at a final concentration of 250 U/mL for 30 min at 37 °C.
5. Centrifuge the digested media at 4000 x g, 4 °C for 30 min and then filter it with a 0.22 µm syringe filter into the pre-wetted regenerated cellulose membrane for 100 kDa.
6. Centrifuge the regenerated cellulose membrane at 4000 x g, 4 °C for 30 min and discard the media.
7. Wash and centrifuge the regenerated cellulose membrane with PBS containing 0.001% Pluronic F-68 three times at 4000 x g, 4 °C for 30 min. Discard the PBS at each step.
8. Collect concentrated AAVs from the top part of the regenerated cellulose membrane and aliquot for further uses.
9. Digest 5 µL of freshly made AAV with DNase I (0.2U/µl) for 15 min at 37 °C for titering. This is followed by 10 min at 95 °C incubation for both inactivating the DNase I and degrading the viral capsids.
10. Prepare a 10-fold serial dilution from digested AAVs using 0.05% Pluronic F-68. Use AAV2-ITR and WPRE primers for titration (Table 1). The titers were calculated by multiplying dd-PCR results with the dilution factors. Results were converted to genome copy/mL (GC/mL).
    NOTE: AAV concentrations for small scale AAV production are expected to be around 1 x 10¹² GC/mL. This protocol is not applicable for AAV strains that do not efficiently release AAVs into media like AAV2¹³.

2. Intravitreal injection of AAV

1. Preparation of equipment
   1. Before starting, prepare 10 µL microsyringe with a 36 G blunt needle. Rinse five times with 70% EtOH, five times in ddH₂O, and finally five times with PBS.
   2. Load the appropriate amount of AAV into the microsyringe for each injection.
   3. Prepare the surgical needle (suture, silk, 6/0), tape, forceps, and scissors.
2. Intravitreal injection
   1. Apply 1 drop of 0.5% tropicamide and 2.5% phenylephrine hydrochloride (e.g., Mydfrin) containing eye drop before anesthesia to dilate pupils.
   2. Anesthetize mice with isoflurane 5% (1 L/min) and continue with 1.5% isoflurane (1 L/min) during the procedure. A small isoflurane chamber is used for the first induction.
   3. Verify the depth of anesthesia by the loss of righting reflex, the withdrawal reflex, and tail pinch response.
   4. Apply 0.3% tobramycin and 0.1% dexamethasone sterile ophthalmic solution to each eye before the injection procedure. Application of eye drop prevents dryness and exerts anti-inflammatory and anti-bacterial effects.
   5. Use the surgical hook to stabilize the upper eyelid. Slightly pull back to expose the dorsal part of the eye. Tape the hook to the bench to hold it in position.
   6. Remove conjunctiva (less than 1 mm²) with the curved iris scissors to expose the sclera of the eye under the dissecting microscope. Use a fresh and sterile insulin syringe (30 G) to puncture the sclera.
   7. Insert the needle of the microsyringe through the same puncture using a micromanipulator. Place the tip of the needle behind the lens in the middle of the eyecup.
   8. Inject 1 µL of AAV2/BP2 and AAV2/PHP.S vectors having 1.63 x 10¹² GC/mL and 1.7 x 10¹² GC/mL concentrations slowly into the vitreous of the eye. Injection volume control is done manually. Leave the needle in place for 1 min and slowly withdraw the needle.
   9. Release the eyelid and apply anti-inflammatory and anti-bacterial topical gel treatment containing 0.3% tobramycin and 0.1% dexamethasone to eyecup. Monitor and evaluate the mice in the following days according to the score sheet in terms of appearance (bright eyes, groomed coat, hunching), behavior (activity, immobilization, self-mutilation), and body weight on a scale of 0 to 3. Each condition has a score from 0-3 and a total score of 3 or above is a termination criterion.
   10. Place the animals in the cage after recovery.

3. Fluorescence and fundus imaging

1. Strain the mouse and dilate the pupil by placing drops of 0.5% tropicamide and 2.5% phenylephrine hydrochloride into each eye.
2. Anesthetize the animal with the above procedure with isoflurane. Assess the depth of anesthesia carefully by pinching the paws.
3. Place the mouse on the imaging stage and verify the proper dilation by checking through the fundus camera. Apply topical gel (0.2% carbomer 980) onto the surface of the eyes to protect eyes from dehydrating under anesthesia and to use it as a coupling gel for imaging.
4. Manipulate the imaging stage to line up with the nosepiece of the objective. Adjust the stage as needed to center the mouse eye. Once the eye is centered, slowly move the objective until it makes contact with the eye.
5. Perform fundus and fluorescence imaging on both eyes to follow up tdTomato expression at 1 week and 2 weeks after intravitreal injection (Figure 3B)¹⁴. Take fundus and fluorescence images at identical settings for comparison of reporter expression.

4. Retina isolation

1. Euthanize animals after fundus and fluorescence imaging by CO₂ inhalation for retina collection at 2 weeks time point.
2. Shift the eyeball forward with the help of a 13.5 cm splitter forceps.
3. Remove the lens together with the leftover vitreous by applying gentle pressure with the forceps.
4. Cut the connection of the eyeball to the optic nerve with precision curved forceps and gently squeeze the retina with the same curved forceps.
5. Transfer dissected retinas into a microcentrifuge tube.
6. Snap freeze retina by placing the tubes into the liquid nitrogen.

5. Tissue genomic DNA isolation

1. Isolate genomic DNA with a commercialized Proteinase K digestion-based tissue genomic DNA kit.
2. Digest retina at 55 °C, 200 x g for 30 min with Proteinase K, which is already supplied in the kit.
3. Perform RNase incubation step, wash steps with wash buffer I and II, and finally elution step according to the manufacturer's protocol.
4. Add an extra spin step after the last wash to remove residual ethanol.
5. Measure the concentration of genomic DNA and store samples at -20 °C.

6. Droplet digital PCR analysis of mouse retina samples for quantification of viral genomes

1. Dilute genomic DNAs from injected retinas in 0.05% Pluronic F-68 solution to reach a final concentration of 1 ng/µL for each sample.
2. Perform separate reactions for WPRE and 18S using target specific primers with a final concentration of 125 nM for each primer.
3. Perform droplet generation in a 20 µL of PCR reaction mix including 1 ng of genomic DNA, 125 nM primers, and 2x evagreen supermix.
4. Load the sample mix in the middle part of the cartridge for droplet generation.
5. After sample loading to cartridge was done, add 70 µL of the droplet generation oil into the bottom row of the cartridge.
6. Put the gasket on top of the cartridge and place it into the droplet generator. Make sure that there is no gap between the gasket and the cartridge.
7. Gently take approximately 40 µL of droplets that are generated in the top well. Add the droplet solution to the semi-skirted 96-well PCR reaction plate.
8. Seal the PCR plate with PCR plate sealer by using aluminum sealing foil.
9. Place PCR plate into 96-well heat-sealed thermal cycler. Use the PCR protocol; 95 °C for 5 min, 40 cycles of 95 °C for 30 s, 60 °C for 1 min, 4 °C for 5 min, 90 °C for 5 min and 4 °C infinite hold. Apply 2 °C/s ramp rate at each cycle to ensure that the droplets reach the correct temperature for each step during the cycling
10. Place PCR plate into the droplet reader to quantify droplets using dd-PCR software. A template is set up using specific settings for evagreen.
11. Analyze data using a 1-D plot graph with each specimen for fluorescence intensity vs droplet number. The threshold value is arranged so that the droplets above the threshold are assigned as positive and the below ones as negative. This is followed by the application of the Poisson algorithm to determine the starting concentration of target DNA in units of copies/µL. The ratio of WPRE versus 18S is calculated for measuring AAV transduction efficiency.