We would like to thank Oezkan Keles, Josephine J&#252;ttner, and Prof. Botond Roska, Institute of Molecular and Clinical Ophthalmology Basel, Complex Viruses Platform for their help and support for AAV production. We also would like to thank Prof. Jean Bennett, Perelman School of Medicine, the University of Pennsylvania for the AAV8/BP2 strain. Animal work is performed at the Gebze Technical University animal facility. For that, we thank Leyla Dikmetas and Prof. Uygar Halis Tazebay for technical assistance and support for animal husbandry. We also would like to thank Dr. Fatma Ozdemir for her comments on the manuscript. This work is supported by TUBITAK, grant numbers 118C226 and 121N275, and Sabanci University Integration grant.

All experimental protocols were accepted by the Sabanci University ethics committee and experiments were conducted in accordance with the statement of &#39;The Association for Research in Vision and Ophthalmology&#39; for the use of animals in research
1. Small scale AAV production12
<ol>
	<li>Culture HEK293T cells using 15 cm plates in complete 10 mL of DMEM/10% FBS until 70-80% confluency.</li>
	<li>Prepare the transfection mixture with 20 &#181;g o...

<ol>
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Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+AAVs+for+efficient+noninvasive+gene+delivery+to+the+central+and+peripheral+nervous+systems.">Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems.</a> Nature Neuroscience. 20 (8), 1172-1179 (2017).</li><li>Guiet, R., Burri, O., Seitz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open+source+tools+for+biological+image+analysis.">Open source tools for biological image analysis.</a> Methods Molecular Biology. 2040, 23-37 (2019).</li><li>Parks, M. 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K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+safety+evaluation+of+AAV2-sFLT01-+a+gene+therapy+for+age-related+macular+degeneration.">Preclinical safety evaluation of AAV2-sFLT01- a gene therapy for age-related macular degeneration.</a> Molecular Therapy. 19 (2), 326-334 (2011).</li><li>Stieger, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+intact+rAAV+particles+up+to+6+years+after+successful+gene+transfer+in+the+retina+of+dogs+and+primates.">Detection of intact rAAV particles up to 6 years after successful gene transfer in the retina of dogs and primates.</a> Molecular Therapy. 17 (3), 516-523 (2009).</li><li>Gyorgy, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allele-specific+gene+editing+prevents+deafness+in+a+model+of+dominant+progressive+hearing+loss.">Allele-specific gene editing prevents deafness in a model of dominant progressive hearing loss.</a> Nature Medicine. 25 (7), 1123-1130 (2019).</li><li>Cox, D. 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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 transduction12,13. 
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...

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 surface1,2. 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 product5,6,7. 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 DNA6,10. 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96-Well Semi-Skirted ddPCR plates</td>
			<td>BioRad</td>
			<td>12001925</td>
			<td>ddPCR</td>
		</tr>
		<tr>
			<td>Amicon Filter</td>
			<td>Millipore</td>
			<td>UFC910096</td>
			<td>AAV</td>
		</tr>
		<tr>
			<td>C1000 TOUCH 96 DEEP WELLS</td>
			<td>BioRad</td>
			<td>1851197</td>
			<td>ddPCR</td>
		</tr>
		<tr>
			<td>C57BL/6JRj mice strain</td>
			<td>Janvier</td>
			<td>C57BL/6JRj</td>
			<td>Mice</td>
		</tr>
		<tr>
			<td>DG8 gaskets</td>
			<td>BioRad</td>
			<td>1863009</td>
			<td>ddPCR</td>
		</tr>
		<tr>
			<td>DG8 Cartridges</td>
			<td>BioRad</td>
			<td>1864008</td>
			<td>ddPCR</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Lonza</td>
			<td>BE12-604Q</td>
			<td>AAV</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>PAN BIOTECH</td>
			<td>L 1825</td>
			<td>AAV</td>
		</tr>
		<tr>
			<td>Droplet generation oil eva green</td>
			<td>BioRad</td>
			<td>1864006</td>
			<td>ddPCR</td>
		</tr>
		<tr>
			<td>Droplet reader oil</td>
			<td>BioRad</td>
			<td>1863004</td>
			<td>ddPCR</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>PAN BIOTECH</td>
			<td>p30-3306</td>
			<td>AAV</td>
		</tr>
		<tr>
			<td>Foil seals for PX1 PCR Plate sealer</td>
			<td>BioRad</td>
			<td>1814040</td>
			<td>ddPCR</td>
		</tr>
		<tr>
			<td>Insulin Syringes</td>
			<td>BD Medical</td>
			<td>320933</td>
			<td>Intravitreal injection</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>ADEKA <img alt="Equation 2" src="/files/ftp_upload/63038/63038eq02v2.jpg" />LA&#199; SANAY<img alt="Equation 2" src="/files/ftp_upload/63038/63038eq02v2.jpg" /> VE T<img alt="Equation 2" src="/files/ftp_upload/63038/63038eq02v2.jpg" />CARET A.<img alt="Equation 1" src="/files/ftp_upload/63038/63038eq01v2.jpg" />.</td>
			<td>N01AB06</td>
			<td>anesthetic</td>
		</tr>
		<tr>
			<td>Microinjector MM33</td>
			<td>World Precision Instruments</td>
			<td>82-42-101-0000</td>
			<td>Intravitreal injection</td>
		</tr>
		<tr>
			<td>Micron IV</td>
			<td>Phoenix Research Labs</td>
			<td>Micron IV</td>
			<td>Microscopy system based on 3-CCD color camera, frame grabber, and off-the-shelf software enables researchers to image mouse retinas.</td>
		</tr>
		<tr>
			<td>Mydfrin (%2.5 phenylephrine hydrochloride)</td>
			<td>Alcon</td>
			<td>S01FB01</td>
			<td>pupil dilation</td>
		</tr>
		<tr>
			<td>Nanofil Syringe 10 &#956;l</td>
			<td>World Precision Instruments</td>
			<td>NANOFIL</td>
			<td>Intravitreal injection</td>
		</tr>
		<tr>
			<td>Needle RN G36, 25 mm, PST 2</td>
			<td>World Precision Instruments</td>
			<td>NF36BL-2</td>
			<td>Intravitreal injection</td>
		</tr>
		<tr>
			<td>PEI-MAX</td>
			<td>Polyscience</td>
			<td>24765-1</td>
			<td>AAV</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>PAN BIOTECH</td>
			<td>P06-07100</td>
			<td>AAV</td>
		</tr>
		<tr>
			<td>Plasmid pHGT1-Adeno1</td>
			<td>PlasmidFactory</td>
			<td>PF1236</td>
			<td>AAV</td>
		</tr>
		<tr>
			<td>Pluronic F-68</td>
			<td>Gibco</td>
			<td>24040032</td>
			<td>AAV</td>
		</tr>
		<tr>
			<td>PX1 PCR Plate Sealer system</td>
			<td>BioRad</td>
			<td>1814000</td>
			<td>ddPCR</td>
		</tr>
		<tr>
			<td>QX200 ddPCR EvaGreen Supermix</td>
			<td>BioRad</td>
			<td>1864034</td>
			<td>ddPCR</td>
		</tr>
		<tr>
			<td>QX200 Droplet Reader/QX200 Droplet Generator</td>
			<td>BioRad</td>
			<td>1864001</td>
			<td>ddPCR</td>
		</tr>
		<tr>
			<td>SPLITTER FORCEP WATCHER 
			MAKER - LENGTH = 13.5 CM</td>
			<td>endostall medical</td>
			<td>EJN-160-0155</td>
			<td>Retina isolation</td>
		</tr>
		<tr>
			<td>Steril Syringe Filter</td>
			<td>AISIMO</td>
			<td>ASF33PS22S</td>
			<td>AAV</td>
		</tr>
		<tr>
			<td>Tissue Genomic DNA Kit</td>
			<td>EcoSpin</td>
			<td>E1070</td>
			<td>gDNA isolation</td>
		</tr>
		<tr>
			<td>Tobradex (0.3% tobramycin / 0.1% dexamethasone)</td>
			<td>Alcon</td>
			<td>S01CA01</td>
			<td>anti-inflammatory / antibiotic</td>
		</tr>
		<tr>
			<td>Tropamid (% 0.5 tropicamide)</td>
			<td>Bilim <img alt="Equation 2" src="/files/ftp_upload/63038/63038eq02v2.jpg" />la&#231; Sanayi ve Ticaret A.<img alt="Equation 1" src="/files/ftp_upload/63038/63038eq01v2.jpg" />.</td>
			<td>S01FA06</td>
			<td>pupil dilation</td>
		</tr>
		<tr>
			<td>Turbonuclease</td>
			<td>Accelagen</td>
			<td>N0103L</td>
			<td>AAV</td>
		</tr>
		<tr>
			<td>Viscotears (carbomer 2 mg/g)</td>
			<td>Bausch+Lomb</td>
			<td>S01XA20</td>
			<td>lubricant eye drop</td>
		</tr>
	</tbody>
</table>

digital droplet pcr method for the quantification of aav transduction efficiency in murine retina

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.

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...

Watch this Scientific Journal Video about Digital Droplet PCR Method for the Quantification of AAV Transduction Efficiency in Murine Retina at JoVE.com

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

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.

Many retinal cell biology laboratories now routinely use Adeno-associated viruses (AAVs) for gene editing and regulatory applications. The efficiency of ...

In this video, we will show how to quantify AAV transduction in mouse retina using digital droplet PCR method, which is extremely sensitive and does absolute quantification of the target DNA. We have developed this method mainly for big AVV constructs that does not allow us to use a reporter gene like CRISPR based constructs. By using this method, we can simply quantify the transduction rate of AAVs in retina.This method relies on sensitivity and absolute quantification of the digital droplet PCR and yields straight forward results. In this video, we will show how to quantify AAV transduction in mouse retina using digital droplet PCR method, which is extremely sensitive and does absolute quantification of the target DNA. We have developed this method mainly for big area constructs that does not allow us to use a reporter gene like CRISPR based constructs.By using this method, we can simply quantify the transduction rate of AAVs in retina. This method relies on sensitivity and absolute quantification of the two droplet PCR and yields straight forward results. Because of the efficiency of the digital droplet PCR methodology, several laboratories already switch from quantitative real-time PCR to digital droplet PCR or AAV titrating.This method can also provide a basis for mitochondrial DNA quantification in retina, as well as checking the efficiency of the CRISPR based mutation correction for gene therapy in the mouse retina. Prepare transfection mixture with AAV helper plasmid, AAV capsid plasmid, AAV vector, and PI solution in five milliliters demand. Add the five milliliters prepared transfection mixture into culture media.Incubate transfected cells at 37 degrees celsius. Collect the media at 48 to 60 hours post transfection. Digest the media with DNAs at a final concentration of 250 units per milliliter for 30 minutes at 37 degrees Celsius and a centrifuge it at 4, 000 g four degrees Celsius for 30 minutes.Pre-wet the regenerated cellulose membrane with PBS. Filter the media with 0.22 micrometers syringe filter into the pre-wetted regenerated cellulose membrane for 100 kilo Daltons. Centrifuge the regenerated cellulose membrane at 4, 000 G four degrees Celsius for 30 minutes and discard the media.Wash and centrifuge the regenerated cellulose membrane with PBS containing 0.001%pluronic F68 three times at 4000 G four degree Celsius for 30 minutes. Collect concentrated AAVs from the top part of the regenerated cellulose membrane and allocate for further use. Here we showed how to make a smaller scale AAV production.After vetting this protocol, you should be able to make AVVs that is sufficient to see the reporter expression in the retina. You can also follow the protocol for digital PCR to be able to titer the produce AAVs. Apply one drop of 0.5%tropicamide and 2.5%phenylephrine hydrochloride containing eye drop before anesthesia to dilate pupils.Anesthetize mice with isoflurane and verify the reflexes. Apply 0.3%tobramycin and 0.1%dexamethasone sterile ophthalmic solution eye drop to each eye before the injection procedure. 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 band to hold in position. Remove conjunctiva with the curved-iris scissors to expose the sclera of the eye.Use a fresh and sterile 30G insulin syringe to puncture the sclera. Insert the needle of the micro syringe through the same puncture using micro manipulator. Place the tip of the needle behind the lens in the middle of the eye cup.Inject one micro liter of AAV solution slowly into the vitreous of the eye. Leave the needle in place for one minute and slowly withdraw the needle. Apply anti-inflammatory and anti-bacterial topical gel treatment containing 0.3%tobramycin and 0.1%dexamethasone to eye cup.Apply one drop off 0.5%tropicamide. And 2.5%phenylephrine hydrochloride containing eye drop before anesthesia to dilate pupils. Apply carbamide two milligrams per gram topical gel onto the surface of the eyes.Adjust the mouse stand as needed to center the mouse eye. Perform fundus and fluorescence imaging on both eyes to follow TdTomato expression one week and two weeks after intravitreal injection. Euthanize the animals using carbon dioxide inhalation before the procedure.Shift eye ball forward with the help of a 13.5 centimeters splitter forceps. Remove the lens together with the leftover vitreous. Cut the connection of the eyeball to the optic nerve with a precision curved forceps and gently squeeze retina with the same curved forceps.Isolate genomic DNA with a commercialized proteinase K digest film-based tissue genomic DNA kit. Digest retina at 55 degrees Celsius 200 G for 30 minutes with proteinase K.Lyse retina completely and follow the manufacturer'sprotocol. In these protocols, we have shown intravitreal injection fundus florescence imaging, as well as retina and genomic DNA isolation.These are very critical procedures for retina, and by following this protocol, you should be able to inject the produced AAVs and follow up their expression profile. And ladle that transduction efficiency by quantifying AAV genomes in retinal genomic DNA. Here, you can see the basic application of digital droplet PCR, where you can make an absolute quantification of AAV genome that transduce in the retinal cells.Dilute genomic DNAs from injected retinas in 0.05%pluronic F 68 solution to reach a final concentration of one nanogram per microliter for each sample. Perform droplet generation in a 20 microliters PCR reaction mix, including one nanogram genomic DNA 125 nanomolars primers and 2X evergreen super mix. Load sample mix to the middle part of the cartridge for droplet generation.Afterwards add 70 microliters droplet generation oil into the bottom row of cartridge. Put the gasket on top of cartridge. Make sure that there is no gap between the gasket and the cartridge.Then place it into the droplet generator. Gently take approximately 40 microliters of droplets that are generated in the top bell. Add droplet solution to semi skirted 96-Well PCR reaction plate.Seal the PCR plate with a PCR plate sealer by using aluminum sealing foil. Place the PCR plate into the 96-Well heat sealed thermal cycler. Use the ddPCR protocol indicated in the article.Place PCR plate into the droplet reader to analyze data. After watching this video, you should be able to perform basic digital PCR technique. You can also use exerted cells with minor amounts of material, which is ideal for digital PCR technique.However, you should carefully adjust the target DNA amount to not exceed the limit of digital droplet PCR. Here's the flow chart for the experimental procedure. We performed small-scale AAV production, which is followed by AAV titering.Then we performed intravitreal injection. Afterwards, we quantified the fluorescent intensity by fundus fluorescence imaging. Then isolated the mouse retinas and extracted genomic DNA from retinas for ddPCR.Titering AAV is also critical to be able to correctly quantify AAV transduction. ddPCR depends on generation of droplets that has diluted target AAV genom. Representative ddPCR results shows positive and negative droplets for the AVV genome.Positive above the threshold and negative is below. After multiplying with the dilution factors, titer of AAVs were found to be 10 to the 12 in genome copies per milliliter. In order to correctly quantify AAV tranduction efficiency, several retinas were injected.Injected animals were imaged, and fluorescence mean intensity was calculated. At two weeks time point, retinas were isolated. And ddPCR was performed using WPRE and 18 S primers.Fluorescence intensity of AAV injected retinas were correlated with AAV genome calculations, showing the power of the methodology. Here, we have shown several techniques, including small-scale AAV preparation intravitreal injection.

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Research

JoVE Journal

Neuroscience

4.3K ビュー.  Sabanci University. このビデオでは、非常に感度が高く、標的DNAの絶対定量を行うデジタル液滴PCR法を用いて、マウスのAAV伝達を定量化する方法を紹介します。我々は、主にCRISPRベースの構築物のようなレポーター遺伝子を使用することを許可しない大きなAVV構造のためにこの方法を開発しました。この方法を用いることで、単にRetinaでのAAVのトランスダクション率を定量化することがで?...