We thank the imaging platform of the Institut de la Vision. We acknowledge the French Muscular Dystrophy Association (AFM) for a PhD fellowship to O.V. and Allergan INC. This work performed in the frame of the LABEX LIFESENSES [reference ANR-10-LABX-65] was supported by French state funds managed by the ANR. We thank Peggy Barbe, and M&#233;lissa Desrosiers for technical assistance with AAV preparations. We are grateful to St&#233;phane Fouquet for excellent technical assistance in confocal microscopy and his expert input with the interpretation of the results.

All animals used in this study were cared for and handled according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
1. Production of Recombinant AAV (rAAV) by Transient Transfection of HEK-293 Cells17,18
NOTE: See McClure C, JoVE (2011)19.
<ol>
	<li>Purify a large-scale plasmid preparation (at least 1 mg/ml) of the AAV vector plasmids. Use 3 plasmids. The AAV helper plasmid carrying the replication and capsid genes without the AAV inverted terminal repeats ITRs, the transgene expression cassette carrying the AAV ITR&#160;referred to as the transfer plasmid, and the plasmid supplying the adenovirus helper genes E2, E4, and VA RNA genes referred to as pHELPER.</li>
	<li>Transfect 293 cells with these 3 plasmids using polyethyleneimine (or other transfection reagent). Above 80% of transfection efficiency is necessary for good viral yields.</li>
	<li>Purify recombinant AAV from 293 cell lysates on an iodixanol gradient. 15%, 25%, 40%, and 60% iodixanol is used to obtain the gradient. Collect 40% iodixanol fraction containing AAV particles, concentrate the fraction after ultracentrifugation at 500,000 x g for 1 hr and buffer exchange against PBS (phosphate buffered saline)&#160;with 0.001% Pluronic.</li>
	<li>To determine viral genomic titer20, perform a DNAse ProteinaseK digest followed by QPCR. The forward and reverse primers amplify the 62 bp region located in the AAV2 ITR. For the plasmid standard, the probe is labeled with FAM and has a black hole quencher (BHQ).</li>
	<li>Carry out qPCR (quantitative polymerase chain reaction)&#160;&#160;in a final volume of 25 &#956;l using 340 nM reverse ITR primer, 100 nM forward ITR primer, 100 nM AAV2 ITR probe, 0.4 mM dNTPs, 2 mM MgCl2, 1x Platinum Taq Buffer, 1U Platinum Taq and 5 &#956;l template (standard, sample and no template control).</li>
	<li>For the standard, use the plasmid for transfection in 7 x 10-fold serial dilutions (5 &#181;l each of 2 x 109 to 2 x 103 genomes/ml resulting in a final concentration from 1010 to 104 genomes/ml).</li>
	<li>Begin the program by an initial denaturation step at 95 &#176;C for 15 min followed by 40 cycles of denaturation at at 95 &#176;C for 1 min and annealing/extension at 60 &#176;C for 1 min. Store at 4 &#176;C for a short-term storage or at -20 &#176;C for long storage periods.</li>
</ol>
2. Intravitreal Injection of AAV
<ol>
	<li>Anesthetize C57BL6J mice with ketamine (50 mg/kg) and xylazine (10 mg/kg) and verify the loss of righting reflex, the withdrawal reflex and tail pinch response indicating a proper anesthetization. Dilate pupils by corneal application of neosynephrine 5% and mydriaticum 0.5% eye drops. Use vet ointment on eyes to prevent dryness while under anesthesia.</li>
	<li>Pass an ultrafine 30 G disposable needle through the equator and next to the limbus, into the vitreous cavity (see Chiu K, JoVE (2007)21). Inject 1 &#181;l stock containing 1 to 4 x 1011 vp of ShH10-GFP or AAV5-GFP with direct observation of the needle in the center of the vitreous cavity (Figure 1). Use one eye as a control for ILM experiments. Inject both eyes for BRB experiments. Apply an anti-inflammatory and antibacterial topical treatment on injected eyes.</li>
	<li>Dilate pupils with neosynephrine and mydriaticum eye drops for imaging. Perform fundus examinations with an eye fundus camera to follow GFP (Green Fluorescent Protein)&#160;expression 1 week, 2 weeks, 1 month, and 2 months after intravitreal injection (Figure 1). Sacrifice mice by CO2 inhalation 1 to 2 months post-injection.</li>
</ol>
3. Immunohistochemistry
<ol>
	<li>For retinal cryosections (Figure 1), dissect enucleated eyes to remove lens and cornea, and immersion fix in 4% paraformaldehyde for 1 hr.
	<ol>
		<li>Cryoprotect the previously fixed eyes in 10% sucrose for 1 hr at room temperature, 20% sucrose for another hr at room temperature. Cryoprotect in 30% sucrose solution, overnight at 4 &#176;C. Freeze and embed eyecups in embedding resin such as Cryo-matrix. Make 10 &#181;m cryosections and mount on slides specially treated for frozen tissue sections and that improve tissue adherence during staining.</li>
		<li>Permeabilize sections with 0.1% Triton X100 in PBS pH 7.4&#160;for 5 min. Wash 2x in PBS and block for 1 hr at room temperature in PBS with 1% BSA, 0.1% Tween 20.</li>
	</ol>
	</li>
	<li>For retinal flatmounts (Figure 1), fix enucleated eyes in 4% paraformaldehyde for 15 min to facilitate the dissection.
	<ol>
		<li>In 15-30 min, dissect the eyes to remove the cornea and lens. Separate the retina from the retinal pigment epithelium (RPE) and sclera by cutting around the ora serrata and the optic nerve. Immerse in 4% paraformaldehyde for another 30 min to fix the retina and the fluorescent protein in transduced retinal cells (fixation must not exceed 24 hr). Wash 5 min in sterile PBS, pH 7.4.</li>
		<li>Change the PBS to blocking buffer (PBS with 1% BSA, 2% normal goat or donkey serum, 0.5% Triton X-100), and incubate in blocking buffer for either 4 hr at room temperature or 4 &#176;C overnight.</li>
	</ol>
	</li>
	<li>Incubate tissues with primary antibodies in blocking buffer for either 4 hr at room temperature or at 4 &#176;C overnight. Wash 3x with PBS. 
	NOTE: The antibodies used are as follows: anti-laminin (1/1,000) labeling the ILM, anti-rhodopsin clone 4D2 (1/500) labeling rods, anti-glutamine synthetase clone GS-6 (1/1,500) labeling M&#252;ller cells, PNA Lectin (1/40) labeling cones.</li>
	<li>Incubate tissues with 1:500 dilution of alexa fluor conjugated secondary antibodies in blocking buffer for 1 hr (cryosections) or 2 hr (flatmounts) at room temperature and protected from light. Wash 3x with PBS.</li>
	<li>Make relieving cuts to the retina and flatmount it onto a glass slide with either the photoreceptor or retinal ganglion cell (RGC) side facing upwards. Add aqueous mountingmedium, apply coverslip and store at 4 &#176;C.</li>
	<li>Perform confocal microscopy on laser-scanning confocal microscope. Acquire images sequentially, line by line, to reduce excitation and emission crosstalk. Define step size according to the Nyquist&#8211;Shannon sampling theorem. Use exposure settings that minimize oversaturated pixels in the final images. Process 12-bit images with FIJI, project Z-sections on a single plane using maximum intensity under Z-project function and finally convert to 8-bit RGB color mode.</li>
</ol>
4. PCR Analysis of Mouse Blood Samples
<ol>
	<li>Inject 100 &#181;l stock containing 1 to 4 x 1011 particles of an AAV-GFP into the penile vein of anesthetized C57BL6J mice (5% of isoflurane for induction in a close chamber and 2.5% of isoflurane through a nose cone during surgery) using an insulin syringe with an ultra-fine 6 mm needle. This animal will serve as a positive control of circulating AAV particles.</li>
	<li>Sample blood from mouse tail before injection and 3 hr, 24 hr, 2 days, and 3 days after ShH10 intravitreal injection or after intrapenile injection (Figure 1). About 10-20 &#181;l is collected in heparin tubes. Sacrifice mice by CO2 inhalation after completion of the procedure.</li>
	<li>Extract genomic DNA from blood samples using the extraction kit of your choice.</li>
	<li>Perform PCR amplification of genomic DNA. For this protocol use the following primer pair: GFP, sense 5&#8217;-CGACACAATCTGCCCTTTCG-3&#8217;, antisense 5&#8217;-CATGGACGAGCTGTACAAGGGA-3&#8217;. The following PCR program was performed: 95 &#176;C 2 min (1 cycle); 95 &#176;C 45 sec, 55 &#176;C 1 min, 72 &#176;C 45 sec (30 cycles); 72&#176;C 5 min (1 cycle); 4 &#176;C hold.</li>
</ol>
5. Optional Evans Blue Method
NOTE: Quantify vascular permeability by measuring albumin leakage from blood vessels into the retina using the Evans blue method10.
<ol>
	<li>Prepare Evans Blue dye by dissolution in a normal saline solution (6 mg/ml), sonication for 5 min in an ultrasonic cleaner, and filtration through a 5 &#181;m filter.</li>
	<li>Anesthetize C57BL6J mice (isoflurane inhalation, see step 4.1), check the loss of righting reflex, the withdrawal reflex and tail pinch response indicating a proper anesthetization, and inject Evans blue (45 mg/kg) through the penile vein (Figure 1). Check that paws, muzzle, and ears of mice become clearly blue following Evans blue injection, confirming the uptake and distribution of the dye.</li>
	<li>Anesthetize C57BL6J mice 3 hr after injection of the dye with ketamine (50 mg/kg) and xylazine (10 mg/kg) and verify the loss of righting reflex indicating a proper anesthetization. Sample intracardially about 500 &#181;l of blood in heparin tubes and perfuse mice for 2 min via the left ventricle with a citrate buffer (0.05 M, pH 3.5; dissolve 7.8 g of citric acid and 3.8 g of sodium citrate in 1 L of distilled water) pre-warmed to 37 &#176;C to prevent vasoconstriction. Mice are sacrificed via the perfusion.</li>
	<li>After perfusion, enucleate and carefully dissect both eyes under an operating microscope to collect the retinas (see Section 3.2). Dry retinas in a centrifugal evaporator overnight, weigh, and extract the Evans blue dye by incubating retinas with 100 &#956;l of formamide for 18 hr at 65 &#176;C with shaking (100 x g). Centrifuge retina samples in 30K omega filter tubes at 20,798.5 x g for 2 hr at 4 &#176;C and centrifuge blood samples at 20,798.5 x g for 15 min at 4 &#176;C.</li>
	<li>Use both supernatants to measure absorbance. Determine absorbance of each sample by subtracting the absorbance maximum for Evans blue at 620 nm to the absorbance minimum at 740 nm. Calculate Evans blue concentration in plasma and retina from a standard curve of Evans blue in formamide. Express BRB permeability in microliter of Evans blue per gram of dry retina per hour (&#181;l plasma/g retinal dry wt/hr).</li>
</ol>
6. Optional Fluorescein Angiography
NOTE: Leakage from retinal blood vessels in mice can be visualized by intraperitoneal injection of sodium fluorescein.
<ol>
	<li>Anesthetize C57BL6J mice (isoflurane inhalation, see step 4.1) and dilate pupils with eye drops (neosynephrine and mydriaticum). Use vet ointment on eyes to prevent dryness while under anesthesia.</li>
	<li>Inject intraperitoneally 0.01-0.02 ml of 10% sodium fluorescein in 0.1 ml sterile saline for fluorescein angiography.</li>
	<li>Collect images of both eyes after injection using an eye fundus camera. Sacrifice mice by CO2 inhalation after the procedure. 
	NOTE: 20 sec are necessary for the retinal vessels to become visible on angiography. Images have to be captured between 3-10 min maximum after fluorescein injection. The leakage spots (if present) are observable after 2.5 min. At later time points the image quality is lost due to sodium fluorescein diffusing into the vitreous, thus only images shortly obtained after are used for analysis.</li>
</ol>

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The authors have nothing to disclose.

The BRB regulates the exchange of molecules between blood and retina. Its breakdown is associated with various diseases such as diabetic retinopathy or age-related macular degeneration (AMD). We recently showed that in a dystrophin knock-out mouse, which displays permeable BRB, the retina becomes more permissive to gene delivery mediated by adeno-associated viral vectors (AAV). However, despite BRB permeability AAV particles injected intraocularly stay confined to the ocular compartment in this model. Our results indicate that gene therapy for diseases that display permeable BRB does not represent additional risks of systemic side effects. Furthermore, they support the fact that other barriers such as the ILM become compromised during retinal disease allowing better access to viral particles. Our findings lead us to think that AAV encoding reporter genes is an excellent tool to study BRB permeability and ILM integrity in animal models of retinal disease. Particularly, the AAV variant ShH10, which specifically labels M&#252;ller glia can be used as a tool to label retinal glia and report on the state of the retina in response to disease. ShH10 will selectively label M&#252;ller glia in both healthy and diseased retinas. However, both ShH10 and other AAVs will provide more efficient gene delivery in retinas with compromised barriers.
Some critical steps in applying the above-described protocols are (1) developing manual dexterity to perform the safest intravitreal injection, and (2) properly fixing the tissue before and after dissection. In studying retinal barriers, the intravitreal injection should be well executed that is - without touching the lens or the retina. Damaging the lens or detaching the retina influences the BRB permeability23,24. Additionally lens damage impedes fundus imaging. Touching the retina can rupture the ILM and damage the retinal structure. To avoid touching the retina, the experimenter should observe the tip of the Hamilton syringe in the middle of the vitreous cavity, in front of the retina. A non-toxic dye can be included (such as Phenol red or fluorescein) in the viral solution to aid in the observation of the fluid injection into the vitreous. Damage to the retina can be readily observed during fundus imaging following the injection procedure. Another critical step is the fixation of the tissue after sufficient expression levels are reached. After enucleation, eyes must be immediately immersed in fixative buffer and before tissue permeabilization a second fixation is necessary in order to prevent GFP leakage. It can be helpful to slit the cornea before immersing the entire eye in the fixative - this will give the fixative a chance to permeate into the vitreous. This step can increase the overall tissue stability.
Lastly, it is also important to inject enough AAV particles to transduce efficiently the retina and to observe GFP expression on eye fundus images. The optimal amount of AAV particles is about 1010-1011 vg per eye, below 1010 particles the GFP expression is not always observed by fundus imaging.
This technique will not give the possibility to directly observe the ILM or the vasculature beneath the BRB. However it provides a way to examine retinal barriers and their modification in disease states, in a way that was previously not possible using the Evans blue assay and fluorescein angiography. Using AAVs with specific retinal transduction properties, it is possible to get better insight into the state of the retina with respect to the glial cells, their extracellular matrix and ILM. After mastering this technique, one can test the efficacy of therapeutic strategies and their influence on BRB and retinal permeability and go towards a better understanding of disease progression and the possibility to restore normal conditions after treatment in a mouse model of retinal disease.

M&#252;ller cells are the major glial component of the retina. Morphologically, they span the retina radially and their endfeet, in contact with the vitreous, face the ILM and secret components of the latter. The ILM is a basement membrane composed of about ten different extracellular matrix proteins (laminin, agrin, perlecan, nidogen, collagen and several heparin sulfate proteoglycans). During development, its presence is indispensable for retinal histogenesis, navigation of optic axons, and&#160;survival of ganglion cells1-3. However, ILM is unessential in adult retina and can be surgically removed in certain pathologies without causing retinal damage4. In gene therapy, this membrane becomes a physical barrier for efficient transduction of the retina using AAVs by intravitreal injection5.
Through the extensive arborization of their processes, M&#252;ller cells provide nutritional and regulatory support to both retinal neurons and vascular cells. M&#252;ller cells are also involved in the regulation of the retinal homeostasis, in the formation and maintenance of the BRB6. Tight junctions between retinal capillary endothelial cells, M&#252;ller cells, astrocytes and pericytes form the BRB. BRB prevents certain substances from entering the retina.In many diseases like diabetic retinopathy, retinal vein occlusion and respiratory diseases, hypoxia of the retina causes leakage through the BRB7-9. This rupture is associated with an increase in vascular permeability leading to vasogenic edema, retinal detachment and retinal damage.
M&#252;ller cells are tightly associated with blood vessels and the basement membrane, playing an important role in both BRB and ILM integrity. Consequently, labeling M&#252;ller glial cells is particularly relevant to the study of the physical state of these retinal barriers.
Classically, BRB permeability is measured using the Evans blue assay consisting of systemic injection of Evans blue dye, which binds non-covalently to plasma albumin. This assay measures the albumin leakage (protein of intermediate size, ~66 kDa) from blood vessels into the retina (see Protocols Section 5)10. Alternatively, the vascular leakage can be visualized by fluorescence retinal angiography attesting for leakage of fluorescein (small molecule, ~359 Da; see Protocols Section 6)11. Nevertheless, both methods allow evaluation of the BRB permeability to small molecules and proteins but they do not provide information about the ILM integrity.
Hence, to study BRB permeability, we used an AAV based method that gives information on the BRB permeability to larger molecules (e.g., AAV particles, 25 nm diameter). Indeed, our method can detect presence of AAV transgene in the blood, which would suggest that ~25 nm diameter particles would be able to infiltrate into the bloodstream. This method also provides information on the structure of the ILM and extracellular matrix proteins in pathological conditions. Two AAV variants are useful for such study: AAV5 and ShH10. Subretinally injected, AAV5 has a natural tropism for photoreceptors and retinal pigment epithelium12 but it cannot get across to the outer retina when administered into the vitreous in wild-type retinas with intact ILM5,13. ShH10 is an AAV variant that has been engineered to specifically target glial cells over neurons14,15. ShH10 selectively labels M&#252;ller cells in both healthy and diseased retinas with increased efficiency in retinas with compromised barriers16. These viral tools coupled with immuhistochemistry and blood-DNA analysis provide information on the state of retinal barriers and their involvement in disease (Figure 1).

<table><tbody><tr><td>C57BL6J mice strain</td><td>JANVIER LABS</td><td></td><td>mice</td></tr><tr><td>Ketamine 500</td><td>Virbac France</td><td></td><td>anesthetic</td></tr><tr><td>Xylazine Rompun 2%</td><td>Bayer Healthcare</td><td></td><td>anesthetic</td></tr><tr><td>Neosynephrine 5% Faure</td><td>Europhta</td><td></td><td>dilatant</td></tr><tr><td>Mydriaticum 0.5%</td><td>Thea</td><td></td><td>dilatant</td></tr><tr><td>Sterdex</td><td>Novartis</td><td></td><td>anti-inflammatory</td></tr><tr><td>Cryomatrix embedding resin</td><td>Thermo Scientific</td><td>6769006</td><td></td></tr><tr><td>Superfrost Plus Adhesion Slides</td><td>Thermo Scientific</td><td>10143352</td><td>slides</td></tr><tr><td>anti-laminin</td><td>Sigma</td><td>L9393</td><td>antibody</td></tr><tr><td>anti-rhodopsin clone 4D2</td><td>Millipore</td><td>MABN15</td><td>antibody</td></tr><tr><td>anti-glutamine synthetase clone GS-6</td><td>Millipore</td><td>MAB302</td><td>antibody</td></tr><tr><td>Anti-Glial Fibrillary Acidic Protein</td><td>Dako</td><td>334</td><td>antibody</td></tr><tr><td>PNA Lectin</td><td>Invitrogen</td><td>L32459</td><td>probe</td></tr><tr><td>Alexa fluor conjugated secondary antibodies</td><td>Invitrogen</td><td></td><td>antibody</td></tr><tr><td>Fluorsave reagent</td><td>Calbiochem</td><td>345789</td><td>mounting medium</td></tr><tr><td>QIAmp DNA Micro Kit</td><td>QIAGEN</td><td>56304</td><td></td></tr><tr><td>GoTaq DNA polymerase</td><td>Promega</td><td>M3001</td><td></td></tr><tr><td>Evans Blue dye</td><td>Sigma</td><td>E2129</td><td>dye</td></tr><tr><td>5 &amp;micro;m filter</td><td>Millipore</td><td></td><td></td></tr><tr><td>Sodium citrate</td><td>Sigma</td><td>S1804</td><td></td></tr><tr><td>Citric acid</td><td>Sigma</td><td>C1909-2.5KG</td><td></td></tr><tr><td>Formamide spectrophotometric</td><td>Sigma</td><td>295876-2L</td><td></td></tr><tr><td>Fluorescein</td><td>Sigma</td><td>F2456</td><td>dye</td></tr><tr><td>Micron III</td><td>Phoenix Research Labs</td><td></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>Insulin Syringes</td><td>Terumo</td><td>SS30M3109</td><td></td></tr><tr><td>Syringe 10 &amp;micro;l Hamilton</td><td>Dutscher</td><td>74487</td><td>Seringue 1701</td></tr><tr><td>Needle RN G33, 25 mm, PST 2</td><td>Fisher Scientific</td><td>11530332</td><td>Intravitreal Injection</td></tr><tr><td>UltraMicroPump UMP3</td><td>World Precision Instruments</td><td>UMP3</td><td>Versatile injector uses microsyringes to deliver picoliter volumes</td></tr><tr><td>UltraMicroPump (UMP3) (one) with SYS-Micro4 Controller</td><td>UMP3-1</td><td>Digital controller</td><td></td></tr><tr><td>Binocular magnifier SZ76</td><td>ADVILAB</td><td>ADV-76B2</td><td>Zoom 0.66 x 5 x LEDs with stand epi and dia / Retinas dissection</td></tr><tr><td>Spring scissors straight - 8.5 cm</td><td>Bionic France S.a.r.l</td><td>15003-08</td><td>Retinas dissection</td></tr><tr><td>curved Vanna scissor</td><td>15004-08</td><td></td><td></td></tr><tr><td>Pince Dumont 5</td><td>11254-20</td><td></td><td></td></tr><tr><td>Veriti 96-Well Thermal Cycler</td><td>Life technologies</td><td>4375786</td><td>Thermocycler</td></tr><tr><td>Ultrasonic cleaner</td><td>Laboratory Supplies</td><td>G1125P1T</td><td></td></tr><tr><td>Nanosep 30k omega tubes</td><td>VWR</td><td></td><td></td></tr><tr><td>Speedvac</td><td>Fisher Scientific</td><td>SC 110 A</td><td></td></tr><tr><td>Spectrofluorometer</td><td>TECAN</td><td>infinite M1000</td><td></td></tr></tbody></table>

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.

We expect increased retinal transduction of M&#252;ller glial cells using ShH10 if the animal model shows perturbations in the structure of the ILM (Figure 2A-B). For example, we have shown that in absence of Dp71,&#160;ShH10 targets specifically but more efficiently M&#252;ller glial cells by intravitreal injection, indicating increased permeability of the ILM in this mouse line compared to wild-type mice16 (Figure 2C-F).
AAV5 can also be used as an indicator of ILM permeability. AAV5, ineffective by intravitreal injection in wild-type mice, becomes strongly effective in gene delivery to photoreceptors in mice with compromised retinal barriers such as the Dp71-null mice16 or in other retinal degenerations22 (Figure 2G-H). This further confirms the ILM disorganization and potential permeability increase in the extra-cellular matrix surrounding the retinal neurons.
We found that, the BRB breakdown of Dp71-null mice remains selective to AAV particles since no trace of AAV were found in blood samples of intravitreally injected Dp71-null mice16 (Figure 3).
<img alt="Figure 1" src="/files/ftp_upload/52451/52451fig1.jpg" /> 
Figure 1: Schematic representation of the general protocol. After AAV production, particles are injected into the vitreous or into the penile vein to anesthetized adult mice. Fundus images with Micron III camera are performed to follow the GFP expression. The GFP expression pattern is analyzed on retinal cryosections and flatmounted retinas thanks to immunohistochemistry and confocal images. The AAV passage through the BRB is assessed by PCR analysis on blood samples coming from intravitreally or intrapenially injected mice, in order to detect the presence of GFP sequence, indicator of the AAV particles presence into the bloodstream.
<img alt="Figure 2" src="/files/ftp_upload/52451/52451fig2.jpg" /> 
Figure 2: ILM permeability to AAV transduction. Confocal images of flatmounted wild-type and Dp71-null retinas labeled with a pan-laminin antibody (A, B) (scale bar: 50 &#181;m). Fundus images showing GFP expression (C, D). Flatmounted retinas one month after ShH10-GFP intravitreal injection (E, F). Three dimensional reconstitution (E*) of the area indicated by an asterisk in E showing transduced M&#252;ller glial cells in green and astrocytes in red (anti-GFAP antibody). Flatmounted retinas one month after intravitreal injection of AAV5-GFP (G, H) (scale bar: 500 &#181;m). Confocal image of the area indicated by an asterisk in H showing transduced photoreceptors in green and cone outer segments in red (PNA lectin staining) (H*). The left column shows results from wild-type mouse retinas and the middle column shows results from Dp71-null mouse retinas. The schema (I) represents AAV transduction of retina with compromised barriers as Dp71-null retina, through the vitreous. Abbreviations : CV, choroidal vessels; RPE, retinal pigment epithelium; PhR, photoreceptor cells (rods and cones); MGC, M&#252;ller glial cells; HC, horizontal cells; BC, bipolar cells; AC, amacrine cells; M, microglia; EC, endothelial cells; P, pericytes; GC, ganglion cells; A, astrocytes; ILM, inner limiting membrane; V, vitreous; AAV, adeno-associated virus. (Re-print with permission from Vacca et al., Glia (2013)16, #3483740951391).
<img alt="Figure 3" src="/files/ftp_upload/52451/52451fig3.jpg" /> 
Figure 3: BRB permeability to AAV particles. PCR amplification of GFP transgene extracted from mouse blood samples. Mice either injected intravitreally (IVT) with ShH10-GFP or intrapenially (IP) with AAV-GFP at different times after injection. Lane 5-18, odd numbers for wild-type mice and even numbers for Dp71-null mice; lane 2, 1 ng of the AAV plasmid, pTR-SB-smCBA-hGFP; lane 3, water; lanes 5-6, blood DNA before injection; lanes 7-8, 3 hr post-IVT; lanes 9-10, 24 hr post-IVT; lanes 11-12, 24 hr post-IP; lanes 13-14, 48 hr post-IVT; lanes 15-16, 48 hr post-IP; lanes 17-18, 72 hr post-IVT; lanes 19-20, 72 hr post-IP. Lanes 1 and 4, Invitrogen scale ladder (100 bp): 100, 200, 300, 400, 500, 600, 700, 800, 1,000, 1,500, 2,000, 3,000 bp. (Re-print with permission from Vacca et al., Glia (2013)16, #3483740951391).

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Using Adeno-associated Virus as a Tool to Study 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 ...

using-adeno-associated-virus-as-tool-to-study-retinal-barriers

Nanyang Technological University

Research

JoVE Journal

Medicine

15.0K Views.  Sorbonne Universtés, UPMC Univ Paris 06, UMR_S 968. 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.

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

Viral Tracing of Genetically Defined Neural Circuitry

Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus (RABV), and more recently vesicular stomatitis virus (VSV), for studying circuitry, is becoming increasingly popular. These higher throughput methods use viruses that transmit between neurons in either the anterograde or retrograde direction.
Recently, a modified RABV for monosynaptic retrograde tracing was developed. (Figure 1A). In this method, the glycoprotein (G) gene is deleted from the viral genome, and resupplied only in targeted neurons. Infection specificity is achieved by substituting a chimeric G, composed of the extracellular domain of the ASLV-A glycoprotein and the cytoplasmic domain of the RABV-G (A/RG), for the normal RABV-G1. This chimeric G specifically infects cells expressing the TVA receptor1. The gene encoding TVA can been delivered by various methods2-8. Following RABV-G infection of a TVA-expressing neuron, the RABV can transmit to other, synaptically connected neurons in a retrograde direction by nature of its own G which was co-delivered with the TVA receptor. This technique labels a relatively large number of inputs (5-10%)2 onto a defined cell type, providing a sampling of all of the inputs onto a defined starter cell type.
We recently modified this technique to use VSV as a transsynaptic tracer9. VSV has several advantages, including the rapidity of gene expression. Here we detail a new viral tracing system using VSV useful for probing microcircuitry with increased resolution. While the original published strategies by Wickersham et al.4 and Beier et al.9 permit labeling of any neurons that project onto initially-infected TVA-expressing-cells, here VSV was engineered to transmit only to TVA-expressing cells (Figure 1B). The virus is first pseudotyped with RABV-G to permit infection of neurons downstream of TVA-expressing neurons. After infecting this first population of cells, the virus released can only infect TVA-expressing cells. Because the transsynaptic viral spread is limited to TVA-expressing cells, presence of absence of connectivity from defined cell types can be explored with high resolution. An experimental flow chart of these experiments is shown in Figure 2. Here we show a model circuit, that of direction-selectivity in the mouse retina. We examine the connectivity of starburst amacrine cells (SACs) to retinal ganglion cells (RGCs).

A method of tracing synaptically connected neurons is described. We use TVA specificity of an upstream cell to probe whether a cell population of interest receives synaptic input from genetically defined cell types.

Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus ...

Neuroscience

Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in retinopathies using non-viral and viral gene delivery to the retina and retinal pigment epithelium (RPE). Subretinal injection is the best approach to deliver viral vectors directly to RPE cells. Before the clinical trial of a gene therapy, it is inevitable to validate the efficacy of the therapy in animal models of various retinopathies. Thus, subretinal injection in mice becomes a fundamental technique for an ocular gene therapy. In this protocol, we provide the easy and replicable technique for subretinal injection of viral vectors to experimental mice. This technique is modified from the intravitreal injection, which is widely used technique in ophthalmology clinics. The representative results of RPE/choroid/scleral complex flat-mount will help to understand the efficacy of this technique and adjust the volume and titer of viral vectors for the extent of gene transduction.

Subretinal injection is a surgical technique for effective gene delivery to retinal pigment epithelium in the mouse eye. Here we describe an easy and replicable method for subretinal injection of viral vectors to retinal pigment epithelium in experimental mice.

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in ...

An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An inflammatory factor, histamine, is demonstrated to compromise vessel integrity in the cultured retina through positive staining of IgG outside of the blood vessels. The effects of histamine itself and those of candidate drugs for potential treatments, such as lipoxin A4, are assessed using three parameters: blood vessel leakage via IgG immunostaining, activation of M&#252;ller cells via GFAP staining and change in neuronal dendrites through staining for MAP2. Furthermore, the layered organization of the retina allows a detailed analysis of the processes of M&#252;ller and ganglion cells, such as changes in width and continuity. While the data presented is with swine retinal culture, the system is applicable to multiple species. Thus, the model provides a reliable tool to investigate the early effects of compromised retinal vessel integrity on different cell types and also to evaluate potential drug candidates for treatment.

A low-cost, easy-to-use and powerful system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach induced by histamine. Blood vessel leakage, M&#252;ller cell activation and the continuity of neuronal processes are utilized to assess the damage response and its reversal with a potential drug, lipoxin A4.

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An ...

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

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 ...

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.

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 ...

Subconjunctival Administration of Adeno-associated Virus Vectors in Small Animal Models

Ocular diseases include a wide range of inherited genetic and acquired disorders that are appealing targets for local drug delivery due to their relative ease of accessibility via multiple administration routes. Subconjunctival (SCJ) injections offer advantages over other intraocular administration routes as they are simple, safe, and usually performed in an outpatient setting. SCJ injections in small animals usually require the assistance of an operating microscope due to the size of the eye. Previous work has demonstrated that SCJ injection of specific adeno-associated virus (AAV) serotypes is a valid gene delivery strategy for targeted transduction of the ocular surface, eye muscle, cornea, and optic nerve, providing a potential approach for the treatment of many ocular diseases.
Herein, a detailed protocol is presented for SCJ injections in a mouse model using an injection system consisting of a programmable infusion/withdrawal syringe pump (which allows for consistent and precise injection speed and pressure) and a gastight removable syringe coupled with microinjection needles. The injection system is also adaptable for other intraocular administration routes such as intrastromal, intracameral, intravitreal, and subretinal injections in small animals. Although the delivery of adeno-associated viral vectors for ocular gene therapy studies is described, the protocol herein can also be adapted for a variety of ophthalmic solutions in small animal models. The key practical steps in the administration route, setup for the injection platform, preparation of the injection, and tips from direct experience will be discussed in detail. In addition, common validation techniques for AAV delivery confirmation to the desired tissues will also be briefly discussed.

In this manuscript, subconjunctival injection is demonstrated as a valid vector delivery method for ocular tissues in mice using an injection system consisting of an infusion/withdrawal syringe pump and a gastight removable syringe coupled with microinjection needles. This injection system is also adaptable for other intraocular administration routes.

Ocular diseases include a wide range of inherited genetic and acquired disorders that are appealing targets for local drug delivery due to their relative ...

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 ...

AAV Isolation by Double Iodixanol Density Gradient Centrifugation

Suspension Culture Production and Purification of Adeno-Associated Virus by Iodixanol Density Gradient Centrifugation for In Vivo Applications

This protocol describes recombinant adeno-associated virus (rAAV) production and purification by iodixanol density gradient centrifugation, a serotype-agnostic method of purifying AAV first described in 1999. rAAV vectors are widely used in gene therapy applications to deliver transgenes to various human cell types. In this work, the recombinant virus is produced by transfection of Expi293 cells in suspension culture with plasmids encoding the transgene, vector capsid, and adenoviral helper genes. Iodixanol density gradient centrifugation purifies full AAV particles based on particle density. Additionally, three steps are included in this now-ubiquitous methodology in order to increase total virus yield, decrease the risk of precipitation due to contaminating proteins, and further concentrate the final virus product, respectively: precipitation of viral particles from cell media using a solution of polyethylene glycol (PEG) and sodium chloride, the introduction of a second round of iodixanol density gradient centrifugation, and buffer exchange via a centrifugal filter. Using this method, it is possible to consistently achieve titers in the range of 1012 viral particles/mL of exceptional purity for in vivo use.

Author Spotlight: Efficient Adeno-Associated Virus Isolation for Pre-Clinical Applications

Adeno-associated virus is produced in suspension cell culture and purified by double iodixanol density gradient centrifugation. Steps are included to increase total virus yield, decrease the risk of virus precipitation, and further concentrate the final virus product. Expected final titers reach 1012 viral particles/mL and are suitable for pre-clinical in vivo use.

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

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