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

Podsumowanie

This protocol presents how to quantify AAV transduction efficiency in mouse retina using digital droplet PCR (dd-PCR) together with small scale AAV production, intravitreal injection, retinal imaging, and retinal genomic DNA isolation.

Streszczenie

Many retinal cell biology laboratories now routinely use Adeno-associated viruses (AAVs) for gene editing and regulatory applications. The efficiency of AAV transduction is usually critical, which affects the overall experimental outcomes. One of the main determinants for transduction efficiency is the serotype or variant of the AAV vector. Currently, various artificial AAV serotypes and variants are available with different affinities to host cell surface receptors. For retinal gene therapy, this results in varying degrees of transduction efficiencies for different retinal cell types. In addition, the injection route and the quality of AAV production may also affect the retinal AAV transduction efficiencies. Therefore, it is essential to compare the efficiency of different variants, batches, and methodologies. The digital droplet PCR (dd-PCR) method quantifies the nucleic acids with high precision and allows performing absolute quantification of a given target without any standard or a reference. Using dd-PCR, it is also feasible to assess the transduction efficiencies of AAVs by absolute quantification of AAV genome copy numbers within an injected retina. Here, we provide a straightforward method to quantify the transduction rate of AAVs in retinal cells using dd-PCR. With minor modifications, this methodology can also be the basis for the copy number quantification of mitochondrial DNA as well as assessing the efficiency of base editing, critical for several retinal diseases and gene therapy applications.

Wprowadzenie

Adeno associated viruses (AAVs) are now commonly used for a variety of retinal gene therapy studies. AAVs provide a safe and efficient way of gene delivery with less immunogenicity and fewer genome integrations. AAV entry into the target cell occurs through endocytosis, which requires binding of receptors and co-receptors on the cell surface^1,2. Therefore, the transduction efficiency of AAVs for different cell types depends mainly on the capsid and its interactions with the host cell receptors. AAVs have serotypes and each serotype can have distinct cellular/tissue tropisms and transduction efficiencies. There are also artificial AAV serotypes and variants generated by chemical modification of the virus capsid, production of hybrid capsids, peptide insertion, capsid shuffling, directed evolution, and rational mutagenesis³. Even minor changes in amino acid sequence or capsid structure can have an influence on interactions with host cell factors and result in different tropisms⁴. In addition to capsid variants, other factors like injection route and batch-to-batch variation of AAV production can affect the transduction efficiency of AAVs in the neuronal retina. Therefore, reliable methods for the comparison of transduction rates for different variants are necessary.

The majority of the methods for determining AAV transduction efficiency rely on reporter gene expression. These include fluorescent imaging, immunohistochemistry, western blot, or histochemical analysis of the reporter gene product^5,6,7. However, due to the size constraint of AAVs, it is not always feasible to include reporter genes to monitor the transduction efficiency. Using strong promoters like the hybrid CMV enhancer/chicken beta-actin or Woodchuck hepatitis post-transcriptional regulatory element (WPRE) as an mRNA stabilizer sequence further complicates the size problem⁸. Therefore, it will also be beneficial to define the transduction rate of injected AAVs with a more direct methodology.

Digital droplet PCR (dd-PCR) is a powerful technique to quantify target DNA from minute amounts of samples. dd-PCR technology depends on encapsulation of the target DNA and PCR reaction mixture by oil droplets. Each dd-PCR reaction contains thousands of droplets. Each droplet is processed and analyzed as an independent PCR reaction⁹. Analysis of droplets enables calculating the absolute copy number of target DNA molecules in any sample by simply using the Poisson algorithm. Since the transduction efficiency of the AAVs is correlated with the copy number of AAV genomes in the neuronal retina, we used the dd-PCR method to quantify AAV genomes.

Here, we describe a dd-PCR methodology to calculate the transduction efficiency of AAV vectors from retinal genomic DNA^6,10. First, AAVs that express tdTomato reporter were generated using the small scale protocol, and titered by the dd-PCR method¹¹. Secondly, AAVs were intravitreally injected into the neuronal retina. To demonstrate the transduction efficiency, we first quantified tdTomato expression using fluorescent microscopy and ImageJ software. This was followed by the isolation of genomic DNA for the quantification of AAV genomes in injected retinas using dd-PCR. Comparison of tdTomato expression levels with the transduced AAV genomes quantified by the dd-PCR showed that the dd-PCR method accurately quantified the transduction efficiency of AAV vectors. Our protocols demonstrated a detailed description of a dd-PCR based methodology to quantify AAV transduction efficiencies. In this protocol, we also show the absolute number of AAV genomes that are transduced after intravitreal injections by simply using the dilution factor after genomic DNA isolation and the dd-PCR results. Overall, this protocol provides a powerful method, which would be an alternative to reporter expression to quantify transduction efficiencies of AAV vectors in the retina.

Protokół

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.

Wyniki

Small scale AAV production is a fast and efficient method that provides vectors for intravitreal injections (Figure 1). Small scale AAV production usually gives titers within the range of 1 x 10¹² GC/ml which is sufficient to detect reporter expression in the retina (Figure 2). Titering of AAV using dd-PCR gives consistent results. ITR2 and WPRE specific primers are routinely used and the starting concentration of each target molecule was calculated w...

Dyskusje

In this protocol, we generated two AAV vectors that have different capsid proteins and then titered them accordingly. One of the most crucial steps of this protocol is to produce sufficient amounts of AAVs that will yield detectable reporter expression after the transduction^12,13.

Titering of AAVs is also an important factor to adjust dosages of AAV for intravitreal injections. Once these important criteria are achieved, it is feasi...

Materiały

Name	Company	Catalog Number	Comments
96-Well Semi-Skirted ddPCR plates	BioRad	12001925	ddPCR
Amicon Filter	Millipore	UFC910096	AAV
C1000 TOUCH 96 DEEP WELLS	BioRad	1851197	ddPCR
C57BL/6JRj mice strain	Janvier	C57BL/6JRj	Mice
DG8 gaskets	BioRad	1863009	ddPCR
DG8 Cartridges	BioRad	1864008	ddPCR
DMEM	Lonza	BE12-604Q	AAV
DPBS	PAN BIOTECH	L 1825	AAV
Droplet generation oil eva green	BioRad	1864006	ddPCR
Droplet reader oil	BioRad	1863004	ddPCR
FBS	PAN BIOTECH	p30-3306	AAV
Foil seals for PX1 PCR Plate sealer	BioRad	1814040	ddPCR
Insulin Syringes	BD Medical	320933	Intravitreal injection
Isoflurane	ADEKA ILAC SANAYI VE TICARET	N01AB06	anesthetic
Microinjector MM33	World Precision Instruments	82-42-101-0000	Intravitreal injection
Micron IV	Phoenix Research Labs	Micron IV	Microscopy system based on 3-CCD color camera, frame grabber, and off-the-shelf software enables researchers to image mouse retinas.
Mydfrin (%2.5 phenylephrine hydrochloride)	Alcon	S01FB01	pupil dilation
Nanofil Syringe 10 μl	World Precision Instruments	NANOFIL	Intravitreal injection
Needle RN G36, 25 mm, PST 2	World Precision Instruments	NF36BL-2	Intravitreal injection
PEI-MAX	Polyscience	24765-1	AAV
Penicillin-Streptomycin	PAN BIOTECH	P06-07100	AAV
Plasmid pHGT1-Adeno1	PlasmidFactory	PF1236	AAV
Pluronic F-68	Gibco	24040032	AAV
PX1 PCR Plate Sealer system	BioRad	1814000	ddPCR
QX200 ddPCR EvaGreen Supermix	BioRad	1864034	ddPCR
QX200 Droplet Reader/QX200 Droplet Generator	BioRad	1864001	ddPCR
SPLITTER FORCEP WATCHER MAKER - LENGTH = 13.5 CM	endostall medical	EJN-160-0155	Retina isolation
Steril Syringe Filter	AISIMO	ASF33PS22S	AAV
Tissue Genomic DNA Kit	EcoSpin	E1070	gDNA isolation
Tobradex (0.3% tobramycin / 0.1% dexamethasone)	Alcon	S01CA01	anti-inflammatory / antibiotic
Tropamid (% 0.5 tropicamide)	Bilim Ilac Sanayi ve Ticaret AS.	S01FA06	pupil dilation
Turbonuclease	Accelagen	N0103L	AAV
Viscotears (carbomer 2 mg/g)	Bausch+Lomb	S01XA20	lubricant eye drop