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 of helper plasmid (pHGT1-Adeno1), 7 &#181;g of capsid plasmid and 7 &#181;g of AAV2-CBA-tdTomato-WPRE vector and 136 &#181;L of PEI solution (1 mg/mL) in 5 mL of DMEM.</li>
	<li>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 &#176;C.</li>
	<li>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 &#176;C.</li>
	<li>Centrifuge the digested media at 4000 x g, 4 &#176;C for 30 min and then filter it with a 0.22 &#181;m syringe filter into the pre-wetted regenerated cellulose membrane for 100 kDa.</li>
	<li>Centrifuge the regenerated cellulose membrane at 4000 x g, 4 &#176;C for 30 min and discard the media.</li>
	<li>Wash and centrifuge the regenerated cellulose membrane with PBS containing 0.001% Pluronic F-68 three times at 4000 x g, 4 &#176;C for 30 min. Discard the PBS at each step.</li>
	<li>Collect concentrated AAVs from the top part of the regenerated cellulose membrane and aliquot for further uses.</li>
	<li>Digest 5 &#181;L of freshly made AAV with DNase I (0.2U/&#181;l) for 15 min at 37 &#176;C for titering. This is followed by 10 min at 95 &#176;C incubation for both inactivating the DNase I and degrading the viral capsids.</li>
	<li>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.</li>
</ol>
2. Intravitreal injection of AAV
<ol>
	<li>Preparation of equipment
	<ol>
		<li>Before starting, prepare 10 &#181;L microsyringe with a 36 G blunt needle. Rinse five times with 70% EtOH, five times in ddH2O, and finally five times with PBS.</li>
		<li>Load the appropriate amount of AAV into the microsyringe for each injection.</li>
		<li>Prepare the surgical needle (suture, silk, 6/0), tape, forceps, and scissors.</li>
	</ol>
	</li>
	<li>Intravitreal injection
	<ol>
		<li>Apply 1 drop of 0.5% tropicamide and 2.5% phenylephrine hydrochloride (e.g., Mydfrin) containing eye drop before anesthesia to dilate pupils.</li>
		<li>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.</li>
		<li>Verify the depth of anesthesia by the loss of righting reflex, the withdrawal reflex, and tail pinch response.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Inject 1 &#181;L 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.</li>
		<li>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.</li>
		<li>Place the animals in the cage after recovery.</li>
	</ol>
	</li>
</ol>
3. Fluorescence and fundus imaging
<ol>
	<li>Strain the mouse and dilate the pupil by placing drops of 0.5% tropicamide and 2.5% phenylephrine hydrochloride into each eye.</li>
	<li>Anesthetize the animal with the above procedure with isoflurane. Assess the depth of anesthesia carefully by pinching the paws.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
4. Retina isolation
<ol>
	<li>Euthanize animals after fundus and fluorescence imaging by CO2 inhalation for retina collection at 2 weeks time point.</li>
	<li>Shift the eyeball forward with the help of a 13.5 cm splitter forceps.</li>
	<li>Remove the lens together with the leftover vitreous by applying gentle pressure with the forceps.</li>
	<li>Cut the connection of the eyeball to the optic nerve with precision curved forceps and gently squeeze the retina with the same curved forceps.</li>
	<li>Transfer dissected retinas into a microcentrifuge tube.</li>
	<li>Snap freeze retina by placing the tubes into the liquid nitrogen.</li>
</ol>
5. Tissue genomic DNA isolation
<ol>
	<li>Isolate genomic DNA with a commercialized Proteinase K digestion-based tissue genomic DNA kit.</li>
	<li>Digest retina at 55 &#176;C, 200 x g for 30 min with Proteinase K, which is already supplied in the kit.</li>
	<li>Perform RNase incubation step, wash steps with wash buffer I and II, and finally elution step according to the manufacturer&#39;s protocol.</li>
	<li>Add an extra spin step after the last wash to remove residual ethanol.</li>
	<li>Measure the concentration of genomic DNA and store samples at -20 &#176;C.</li>
</ol>
6. Droplet digital PCR analysis of mouse retina samples for quantification of viral genomes
<ol>
	<li>Dilute genomic DNAs from injected retinas in 0.05% Pluronic F-68 solution to reach a final concentration of 1 ng/&#181;L for each sample.</li>
	<li>Perform separate reactions for WPRE and 18S using target specific primers with a final concentration of 125 nM for each primer.</li>
	<li>Perform droplet generation in a 20 &#181;L of PCR reaction mix including 1 ng of genomic DNA, 125 nM primers, and 2x evagreen supermix.</li>
	<li>Load the sample mix in the middle part of the cartridge for droplet generation.</li>
	<li>After sample loading to cartridge was done, add 70 &#181;L of the droplet generation oil into the bottom row of the cartridge.</li>
	<li>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.</li>
	<li>Gently take approximately 40 &#181;L of droplets that are generated in the top well. Add the droplet solution to the semi-skirted 96-well PCR reaction plate.</li>
	<li>Seal the PCR plate with PCR plate sealer by using aluminum sealing foil.</li>
	<li>Place PCR plate into 96-well heat-sealed thermal cycler. Use the PCR protocol; 95 &#176;C for 5 min, 40 cycles of 95 &#176;C for 30 s, 60 &#176;C for 1 min, 4 &#176;C for 5 min, 90 &#176;C for 5 min and 4 &#176;C infinite hold. Apply 2 &#176;C/s ramp rate at each cycle to ensure that the droplets reach the correct temperature for each step during the cycling</li>
	<li>Place PCR plate into the droplet reader to quantify droplets using dd-PCR software. A template is set up using specific settings for evagreen.</li>
	<li>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/&#181;L. The ratio of WPRE versus 18S is calculated for measuring AAV transduction efficiency.</li>
</ol>

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The authors have nothing to disclose.

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><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 ILAC SANAYI VE TICARET</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 &amp;mu;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&lt;br/&gt; 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 Ilac Sanayi ve Ticaret AS.</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...

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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 ...

digital-droplet-pcr-method-for-quantification-aav-transduction

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

4.5K Views.  Sabanci University. 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. 

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

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling M&#252;ller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms.
There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5&#160;has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label M&#252;ller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

Medicine

Purification of AAV Vectors by Single-Step Heparin Affinity Chromatography

Isolation of Adeno-Associated Viral Vectors Through a Single-Step and Semi-Automated Heparin Affinity Chromatography Protocol

Adeno-associated virus (AAV) has become an increasingly valuable vector for in vivo gene delivery and is currently undergoing human clinical trials. However, the commonly used methods to purify AAVs make use of cesium chloride or iodixanol density gradient ultracentrifugation. Despite their advantages, these methods are time-consuming, have limited scalability, and often result in vectors with low purity. To overcome these constraints, researchers are turning their attention to chromatography techniques. Here, we present an optimized heparin-based affinity chromatography protocol that serves as a universal capture step for the purification of AAVs.
This method relies on the intrinsic affinity of AAV serotype 2 (AAV2) for heparan sulfate proteoglycans. Specifically, the protocol entails the co-transfection of plasmids encoding the desired AAV capsid proteins with those of AAV2, yielding mosaic AAV vectors that combine the properties of both parental serotypes. Briefly, after the lysis of producer cells, a mixture containing AAV particles is directly purified following an optimized single-step heparin affinity chromatography protocol using a standard fast protein liquid chromatography (FPLC) system. Purified AAV particles are subsequently concentrated and subjected to comprehensive characterization in terms of purity and biological activity. This protocol offers a simplified and scalable approach that can be performed without the need for ultracentrifugation and gradients, yielding clean and high viral titers.

Author Spotlight: Improved Method for Production and Purification of Adeno-Associated Viral Vectors

This manuscript describes a detailed protocol for the generation and purification of adeno-associated viral vectors using an optimized heparin-based affinity chromatography method. It presents a simple, scalable, and cost-effective approach, eliminating the need for ultracentrifugation. The resulting vectors exhibit high purity and biological activity, proving their value in preclinical studies.

Adeno-associated virus (AAV) has become an increasingly valuable vector for in vivo gene delivery and is currently undergoing human clinical trials. ...

Bioengineering

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

Time-Lapse Imaging of Neuronal Arborization using Sparse Adeno-Associated Virus Labeling of Genetically Targeted Retinal Cell Populations

Discovering mechanisms that pattern dendritic arbors requires methods to visualize, image, and analyze dendrites during development. The mouse retina is a powerful model system for the investigation of cell type-specific mechanisms of neuronal morphogenesis and connectivity. The organization and composition of retinal subtypes are well-defined, and genetic tools are available to access specific types during development. Many retinal cell types also constrain their dendrites and/or axons to narrow layers, which facilitates time-lapse imaging. Mouse retina explant cultures are well suited for live-cell imaging using confocal or multiphoton microscopy, but methods optimized for imaging dendrite dynamics with temporal and structural resolution are lacking. Presented here is a method to sparsely label and image the development of specific retinal populations marked by the Cre-Lox system. Commercially available adeno-associated viruses (AAVs) used here expressed membrane-targeted fluorescent proteins in a Cre-dependent manner. Intraocular delivery of AAVs in neonatal mice produces fluorescent labeling of targeted cell types by 4-5 days post-injection (dpi). The membrane fluorescent signals are detectable by confocal imaging and resolve fine branch structures and dynamics. High-quality videos spanning 2-4 h are acquired from imaging retinal flat-mounts perfused with oxygenated artificial cerebrospinal fluid (aCSF). Also provided is an image postprocessing pipeline for deconvolution and three-dimensional (3D) drift correction. This protocol can be used to capture several cellular behaviors in the intact retina and to identify novel factors controlling neurite morphogenesis. Many developmental strategies learned in the retina will be relevant for understanding the formation of neural circuits elsewhere in the central nervous system.

Here, we present a method for investigating neurite morphogenesis in postnatal mouse retinal explants by time-lapse confocal microscopy. We describe an approach for sparse labeling and acquisition of retinal cell types and their fine processes using recombinant adeno-associated virus vectors that express membrane-targeted fluorescent proteins in a Cre-dependent manner.

Discovering mechanisms that pattern dendritic arbors requires methods to visualize, image, and analyze dendrites during development. The mouse retina is a ...

Standardized Protocol for Viral Genome Quantification Using Digital Droplet PCR

Quantification of Adeno-Associated Viral Genomes in Purified Vector Samples by Digital Droplet Polymerase Chain Reaction

Adeno-associated virus (AAV) is a non-pathogenic virus used as a delivery vehicle to transfer therapeutic genes into patients. Accurate quantification of AAV genome copy number in vector preparations is essential for bioprocess optimization and dosage calculation in both preclinical and clinical studies of AAV-based gene therapy products. Currently, a consensus protocol for AAV viral genome titration is lacking. Here, we present a digital droplet PCR (dd_PCR) protocol for the quantification of viral genomes in purified vector samples. Samples are treated with DNase I to eliminate unpackaged contaminant DNA. DNase-treated samples are then mixed with an appropriate primer-probe set (designed according to the target AAV genome) and PCR reagents, and then loaded into a droplet generator. The prepared droplets are transferred into a PCR plate, where PCR amplification is performed and analyzed. The viral genome titer is calculated based on the concentration (copies/&#181;L), accounting for sample dilutions. A successful measurement shows a clear separation of the positive and negative droplet clouds, has at least 10,000 accepted droplets, shows a value between 10 copies/&#181;L and 10,000 copies/&#181;L, and has a coefficient of variation (CV) between repeats lower than 20%. Reliable viral genome titration will aid in the development of safe and effective AAV-based gene therapy products.

Author Spotlight: Optimizing Digital Droplet PCR Method for Accurate Adeno-Associated Viral Genome Quantification

Precise quantification of adeno-associated virus (AAV) vector genome copies is critical, but a standardized protocol has yet to be established. This protocol describes a validated method for preparing purified AAV samples and conducting digital droplet polymerase chain reaction (dd_PCR) to reliably quantify the viral genome titer.

Adeno-associated virus (AAV) is a non-pathogenic virus used as a delivery vehicle to transfer therapeutic genes into patients. Accurate quantification of ...

A Quantitative Dot Blot Assay for AAV Titration and Its Use for Functional Assessment of the Adeno-associated Virus Assembly-activating Proteins

While adeno-associated virus (AAV) is widely accepted as an attractive vector for gene therapy, it also serves as a model virus for understanding virus biology. In the latter respect, the recent discovery of a non-structural AAV protein, termed assembly-activating protein (AAP), has shed new light on the processes involved in assembly of the viral capsid VP proteins into a capsid. Although many AAV serotypes require AAP for assembly, we have recently reported that AAV4, 5, and 11 are exceptions to this rule. Furthermore, we demonstrated that AAPs and assembled capsids of different serotypes localize to different subcellular compartments. This unexpected heterogeneity in the biological properties and functional roles of AAPs among different AAV serotypes underscores the importance of studies on AAPs derived from diverse serotypes. This manuscript details a straightforward dot blot assay for AAV quantitation and its application to assess AAP dependency and serotype specificity in capsid assembly. To demonstrate the utility of this dot blot assay, we set out to characterize capsid assembly and AAP dependency of Snake AAV, a previously uncharacterized reptile AAV, as well as AAV5 and AAV9, which have previously been shown to be AAP-independent and AAP-dependent serotypes, respectively. The assay revealed that Snake AAV capsid assembly requires Snake AAP and cannot be promoted by AAPs from AAV5 and AAV9. The assay also showed that, unlike many of the common serotype AAPs that promote heterologous capsid assembly by cross-complementation, Snake AAP does not promote assembly of AAV9 capsids. In addition, we show that the choice of nuclease significantly affects the readout of the dot blot assay, and thus, choosing an optimal enzyme is critical for successful assessment of AAV titers.

This manuscript details a straightforward dot blot assay for quantitation of adeno-associated virus (AAV) titers and its application to study the role of assembly-activating proteins (AAPs), a novel class of non-structural viral proteins found in all AAV serotypes, in promoting the assembly of capsids derived from cognate and heterologous AAV serotypes.

While adeno-associated virus (AAV) is widely accepted as an attractive vector for gene therapy, it also serves as a model virus for understanding virus ...

Biology

Longitudinal In Vivo Imaging of Retinal Cells with Tailored Adeno-Associated Virus Serotypes via Confocal Scanning Laser Ophthalmoscopy

The dynamic nature of retinal cellular processes necessitates advancements in gene delivery and live monitoring techniques to enhance the understanding and treatment of ocular diseases. This study introduces an optimized adeno-associated virus (AAV) approach, utilizing specific serotypes and promoters to achieve optimal transfection efficiency in targeted retinal cells, including retinal ganglion cells (RGCs) and M&#252;ller glia. Leveraging the precision of confocal scanning laser ophthalmoscopy (CSLO), this work presents a non-invasive method for in vivo imaging that captures the longitudinal expression of AAV-mediated green fluorescent protein (GFP). This approach eliminates the need for terminal procedures, preserving the continuity of observation and the well-being of the subject. Furthermore, the GFP signal can be traced in AAV-infected RGCs along the visual pathway to the superior colliculus (SC) and lateral geniculate nucleus (LGN), enabling the potential for direct visual pathway mapping. These findings provide a detailed protocol and demonstrate the application of this powerful tool for real-time studies of retinal cell behavior, disease pathogenesis, and the efficacy of gene therapy interventions, offering valuable insights into the living retina and its connections.

This protocol describes the use of adeno-associated virus (AAV) vectors for cell-specific labeling and in vivo imaging using a confocal scanning laser ophthalmoscope (CSLO). This method enables the investigation of different retinal cell types and their contributions to retinal function and disease.

The dynamic nature of retinal cellular processes necessitates advancements in gene delivery and live monitoring techniques to enhance the understanding ...

Measurement of Mitochondrial Membrane Potential In Vivo using a Genetically Encoded Voltage Indicator

Mitochondrial membrane potential (MMP, &#916;&#936;m) is critical for mitochondrial functions, including ATP synthesis, ion transport, reactive oxygen species (ROS) generation, and the import of proteins encoded by the nucleus. Existing methods for measuring &#916;&#936;m typically use lipophilic cation dyes, such as Rhodamine 800 and tetramethylrhodamine methyl ester (TMRM), but these are limited by low specificity and are not well-suited for in vivo applications. To address these limitations, we have developed a novel protocol utilizing genetically encoded voltage indicators (GEVIs). Genetically encoded voltage indicators (GEVIs), which generate fluorescent signals in response to membrane potential changes, have demonstrated significant potential for monitoring plasma membrane and neuronal potentials. However, their application to mitochondrial membranes remains unexplored. Here, we developed protein-based mitochondrial-targeted GEVIs capable of detecting &#916;&#936;m fluctuations in cells and the motor cortex of living animals. The mitochondrial potential indicator (MPI)offers a non-invasive approach to study &#916;&#936;m dynamics in real-time, providing a method to investigate mitochondrial function under both normal and pathological conditions.

This protocol describes the application of mitochondria-targeted genetically encoded voltage indicators (GEVIs). These GEVIs offer a significant advantage over traditional mitochondrial membrane potential dyes by enabling specific, in vivo, and real-time monitoring of mitochondrial membrane potential.

Mitochondrial membrane potential (MMP, &#916;&#936;m) is critical for mitochondrial functions, including ATP synthesis, ion transport, reactive oxygen ...

AAV-Based Therapies via Intravenous Injection in Preclinical Mouse Models

Consistent Delivery of Adeno-Associated Virus via Lateral Tail-Vein Injection in Adult Mice

Many disorders affect multiple organs or involve different regions of the body, so it is critical to deliver therapeutics systemically to target the affected cells located in different sites. Intravenous injection is a widely used systemic delivery route in preclinical studies that assess treatments intended for body-wide administration. In adult mice, it involves the intravenous administration of the therapeutic agent into the mouse&#39;s lateral tail veins. When mastered, tail-vein injections are safe and fast, and only require simple and commonly available tools. However, tail-vein injections are technically challenging and require extensive training and continuous practice to ensure the accurate delivery of the intended dose.
Here we describe a detailed, optimized, lateral tail-vein injection protocol that we have developed based on our experience and on recommendations that had been previously reported by other groups. Other than the mouse restrainers and insulin syringes, this protocol requires only reagents and equipment that are readily available in most labs. We found that following this protocol results in consistently successful intravenous delivery of adeno-associated virus (AAV) into the tail veins of unsedated 7-9 week-old mice. Additionally, we describe the optimized protocols for the histological detection of fluorescent reporter proteins and vector genome per diploid genome (vg/dg) quantification used to assess AAV transduction and biodistribution. The goal of this protocol is to aid experimenters in easily performing tail-vein injections successfully and consistently, which can reduce the practice time needed to master the technique.

Author Spotlight: Developing Gene Therapies for Muscular Dystrophies and Neuromuscular Disorders

Here we detail an optimized protocol for mouse lateral tail-vein injection to systemically administer adeno-associated virus (AAV) in adult mice. Additionally, we describe protocols of commonly used assays to assess AAV transduction.

Many disorders affect multiple organs or involve different regions of the body, so it is critical to deliver therapeutics systemically to target the ...

A Step-By-Step Method to Detect Neutralizing Antibodies Against AAV using a Colorimetric Cell-Based Assay

Recombinant adeno-associated viruses (rAAV) have proven to be a safe and successful vector for transferring genetic material to treat various health conditions in both the laboratory and the clinic. However, pre-existing neutralizing antibodies (NAbs) against AAV capsids pose an ongoing challenge for the successful administration of gene therapies in both large animal experimental models and human populations. Preliminary screening for host immunity against AAV is necessary to ensure the efficacy of AAV-based gene therapies as both a research tool and as a clinically viable therapeutic agent. This protocol describes a colorimetric in vitro assay to detect neutralizing factors against AAV serotype 6 (AAV6). The assay utilizes the reaction between an AAV encoding an alkaline phosphatase (AP) reporter gene and its substrate NBT/BCIP, which generates an insoluble quantifiable purple stain upon combination. 
In this protocol, serum samples are combined with an AAV expressing AP and incubated to permit potential neutralizing activity to occur. Virus serum mixture is subsequently added to cells to allow for viral transduction of any AAVs that have not been neutralized. The NBT/BCIP substrate is added and undergoes a chromogenic reaction, corresponding to viral transduction and neutralizing activity. The proportion of area colored is quantitated using a free software tool to generate neutralizing titers. This assay displays a strong positive correlation between coloration and viral concentration. Assessment of serum samples from sheep before and after administration of a recombinant AAV6 led to a dramatic increase in neutralizing activity (125 to &gt;10,000-fold increase). The assay displayed adequate sensitivity to detect neutralizing activity in &gt;1:32,000 serum dilutions. This assay provides a simple, rapid, and cost-effective method to detect NAbs against AAVs.

A comprehensive laboratory protocol and analysis workflow are described for a rapid, cost-effective, and straightforward colorimetric cell-based assay to detect neutralizing elements against AAV6.

Recombinant adeno-associated viruses (rAAV) have proven to be a safe and successful vector for transferring genetic material to treat various health ...