Name: limbal approach-subretinal injection of viral vectors for gene therapy in mice retinal pigment epithelium
Uploaded: 2015-08-07T14:00:00.000+00:00
Duration: 6 min 48 s
Description: Watch this Scientific Journal Video about Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium at JoVE.com

This study was supported by the Seoul National University Research Grant (800-20140542), the Pioneer Research Program of NRF/MEST (2012-0009544), and the Bio-Signal Analysis Technology Innovation Program of NRF/MEST (2009-0090895),&#160;and&#160;the Grant of NRF/MEST (2015M3A9E6028949).

All experiments on animals were performed in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research, and the guidelines and regulations set forth by the Seoul National University Institutional Animal Care and Use Committee and Seoul National University Hospital Biosafety Committee.
1. Preparing Injection Kit and Viral Vectors
<ol>
	<li>Prepare the microliter syringe equipped with a 33 G blunt needle sterilized using ethylene oxide gas. Dilute the viral vectors in PBS for the adequate titer in the micro tube (i.e., 1 x 106 TU/&#181;l). Flush the syringe several times with viral vectors to remove any dead space in the syringe.</li>
</ol>
2. Subretinal Injection of Viral Vectors
<ol>
	<li>Anesthetize the adult mice (i.e., 6 - 8 weeks old) with an intraperitoneal injection of the mixture of tiletamine and zolazepam (1:1, 2.25 mg/kg body weight) and xylazine hydrochloride (0.7 mg/kg body weight) or an alternative suitable anesthesia regime.</li>
	<li>Dilate pupils with an eye drop of phenylephrine 0.5% and tropicamide 0.5%.</li>
	<li>Prepare the microliter syringe by loading with 1.5 - 2 &#181;l of viral vectors.</li>
	<li>Open the eyelid and protrude the eye to expose the equator for convenient injection and focus on under the operating microscope. Maintain the protruded eye position until finishing the injection, or displacement of the needle can occur during the injection. To hold the eyeball firmly, place the fingers outside the orbital rim.</li>
	<li>Apply a drop of ophthalmic viscoelastic solution to the corneal surface.</li>
	<li>Place a small round cover slide on the top of the cornea to visualize the retina.</li>
	<li>Puncture a small hole at slight posterior to the limbus using a sterile 30&#160;G 1/2 needle for the further subretinal injection. Make the hole inferior for the right eye, and superior for the left eye for the convenience. 
	NOTE: If the hole is made at temporal or nasal, it&#8217;s hard to be covered by the eyelid after the injection. &#160;Initial puncture should be made slightly posterior to the limbus to avoid the limbal vessels which run along the limbus and can be easily recognized.&#160;&#160;Be careful to avoid hitting the lens with needle while making the initial puncture. Do not insert the entire bevel of the needle to avoid lens puncture.</li>
	<li>Place the 33 G blunt needle of microliter syringe through the pre-punctured hole and approach the needle into the subretinal space until the point when mild resistance is felt. 
	NOTE: For the subretinal injection, the best approach angle of the needle is about 45 degrees against iris plane, and the blunt needle should be pushed posteriorly toward peripapillary area. Dotted square indicated the suggested needle pathway across the vitreous cavity for the subretinal injection in Figure 1. 
	NOTE: There will be no resistance felt when piercing (or passing) the retinal layers as they are very soft. Thus, the first feeling of mild resistance indicates that the needle has already touched the RPE layer and the insertion of the needle should be stopped. Be careful not to penetrate scleral tissue with excessive pressure because the needle should be placed in the potential subretinal space. If the needle punctures the scleral tissue by excessive power, it enters into the extraocular orbital spaces without resistance. (This step is critical)</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/53030/53030fig1.jpg" /> 
Figure 1.&#160;Schematic Diagram of the Subretinal Injection.&#160;H&#38;E stained cross section of the mouse eye depicting the structures with a needle pathway for the subretinal injection marked (a dotted square). Magnification:40X, Scale bar: 500 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53030/53030fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="9">
	<li>Inject the viral vectors (i.e.,&#160;1 x 106 TU/&#181;l) gently into the subretinal space without tremor to avoid unwanted tissue damage and withdraw the needle gently. Hold the eyeball firmly during the injection as described in 2.4.</li>
	<li>Observe the formation of subretinal bleb after the injection under operating microscope to make sure there is no retinal bleeding.</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/53030/53030fig2.jpg" /> 
Figure 2. Subretinal Belb Formation without Retinal Hemorrhage.&#160;Microscopic view of subretinal belb after the subretinal injection under operating microscope shows successful bleb formation without retinal hemorrhage. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53030/53030fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="11">
	<li>Gently close the eyelid to cover the injection site for self-sealing.&#160;Return the mice to home cage and keep alive until the evaluation. 
	NOTE: After the researchers get used to the procedures, they can get the repeatable results with rapid procedures by skipping these steps (2.5, 6, and 10)</li>
</ol>
3. Evaluation of the Efficacy of Gene Delivery in Retinal Pigment Epithelium
<ol start="1">
	<li>Sacrifice the mice in the CO2 chamber. Enucleate the eyes using scissors.</li>
	<li>Fix the eyes in the 4% paraformaldehyde solution at 4&#160;&#176;C&#160;for a minimum 1 hr&#160;to few days.</li>
	<li>Grab the cornea with fine forceps and puncture the cornea with micro-scissors. Remove the cornea after scissoring along the limbus. Remove the lens with forceps.</li>
	<li>Trim the ciliary body with micro-scissors allowing retinal detachment. Gently pull the whole retina from the RPE/choroid/sclera complex. Cut the RPE/choroid/scleral complex radially from the periphery to the center near optic nerve several times to make 4 to 8 leaves for flat mounting.</li>
	<li>Flip the RPE/choroid/scleral complex to make RPE side down, and trim all the remaining tissues at the scleral side including muscles, conjunctiva and optic nerve. This step is important to make a flat RPE/choroid/scleral complex because any remaining tissue makes the complex uneven.</li>
	<li>Incubate the complex with primary and secondary antibody for your specific research purposes (optional, see the references for detailed methods). Flat-mount the RPE/choroid/scleral complexes on the glass slide and absorb the PBS with an absorbent sponge.</li>
	<li>Add 30 &#181;l&#160;of mounting solution and place the cover slide. Observe the sample for the efficacy of vector delivery under fluorescein microscope. Keep the samples in the refrigerator for further observation.</li>
</ol>

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A., Roska, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+for+blindness.">Gene therapy for blindness.</a> Annu Rev Neurosci. 36, 467-488 (2013).</li><li>Allocca, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+adeno-associated+virus+serotypes+efficiently+transduce+murine+photoreceptors.">Novel adeno-associated virus serotypes efficiently transduce murine photoreceptors.</a> J Virol. 81 (20), 11372-11380 (2007).</li><li>Takahashi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+transduction+of+ocular+cells+with+a+bovine+immunodeficiency+viral+vector.">Sustained transduction of ocular cells with a bovine immunodeficiency viral vector.</a> Hum Gene Ther. 13 (11), 1305-1316 (2002).</li><li>Rolling, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapeutic+prospects+in+early+onset+of+severe+retinal+dystrophy:+restoration+of+vision+in+RPE65+Briard+dogs+using+an+AAV+serotype+4+vector+that+specifically+targets+the+retinal+pigmented+epithelium.">Gene therapeutic prospects in early onset of severe retinal dystrophy: restoration of vision in RPE65 Briard dogs using an AAV serotype 4 vector that specifically targets the retinal pigmented epithelium.</a> Bull Mem Acad R Med Belg. 161 (10-12), 497-508 (2006).</li><li>Le Meur, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+of+vision+in+RPE65-deficient+Briard+dogs+using+an+AAV+serotype+4+vector+that+specifically+targets+the+retinal+pigmented+epithelium.">Restoration of vision in RPE65-deficient Briard dogs using an AAV serotype 4 vector that specifically targets the retinal pigmented epithelium.</a> Gene Ther. 14 (4), 292-303 (2007).</li><li>Alexander, J. 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The authors have nothing to disclose.

In this video article, we described the limbal-approach subretinal injection technique in detail with representative results of RPE/choroid/scleral flat-mount. This is an easy and convenient technique for subretinal injection of viral vectors into RPE. Direct visualization of bleb formation during the injection is an important step for accurate delivery for the beginners. There are some subretinal injection techniques introduced in Journal of Visualized Experiments8-10. Subretinal space is the potential space between retina and RPE, therefore there are two possible routes to approach the subretinal space. One is an inside approach to the subretinal space via vitreous and retina and the other is an outside approach via RPE and sclera. This limbal-approach subretinal injection technique is the former one via intravitreal space and retina. This technique has an advantage to provide more posterior bleb formation compared to the latter because the latter approach makes a bleb from the peripheral side via scleral tunneling or incision, which should be made over the peripheral retina. In addition, simple limbal puncture is easier than scleral tunneling without retinal penetration. For the neonatal mice, however, it would be more appropriate to use the outside approach9. Because neonatal mouse&#8217;s eyeball is very small and full of the lens, limbal-approach technique can cause unwanted cataract formation or even phthisis bulbi.
There are some critical steps in this protocol. First of all, initial puncture should be place slightly posterior to the limbus because too anterior puncture at the cornea makes iris incarceration, or too posterior puncture makes direct hole at the peripheral retina. Secondly, it is recommended to be careful to avoid hitting the lens with needle while making the initial puncture. It is better not to insert the entire bevel of the needle to avoid lens puncture. Thirdly, the best approach angle of the needle is about 45 degrees against iris plane, and the blunt needle should be pushed posteriorly toward peripapillary area. Finally, the needle should be placed in the subretinal space during the injection, resulting in uniform bleb formations, otherwise the displaced needle causes intravitreal injection.
The eye is a small organ with unique features that make it an attractive target for gene therapy11. Specifically, it is a small-enclosed compartment with immune-privilege that enables gene therapy with relatively low toxicity and immune reactions in the eye and adverse systemic effects. Easy visualization and ready accessibility to surgical approaches are other advantages of the eye for gene therapy. The retina has a highly organized laminar structure with 10 distinct layers. Thus, accurate spatial gene delivery can only be achieved by the appropriate methods of intraocular injection. The two most common methods for intraocular delivery of viral vectors are intravitreal injection and subretinal injection6. In general, intravitreal injection is the preferred route for inner retina targeting retinal ganglion cells and M&#252;ller cells. Subretinal injection is the preferred route for outer retina targeting photoreceptor cells and RPE cells. For an efficient transduction in RPE, it is necessary to deliver viral vectors subretinally despite the potential for damage to the retina.
In this study, we used LV vectors with GFP and RFP for the visualization. Because viral vectors have tropism for specific cell types of the retina, viral vectors should be chosen according to tropism of AAV, LV, and Ad to target cells5. For example, AAV2/2, AAV2/6 and AAV2/8 are the highest transduction efficiency to RGC and M&#252;ller cells. AAV2/5, 2/7, 2/8 and 2/9 efficiently transduce photoreceptor cells and RPE with AAV2/8 being the most efficient serotype in mice5,12. AAV2/1, AAV2/2, AAV2/4, AAV2/6, and AAV2/8 efficiently transduce RPE5. For LV vectors, there is a high-level of expression in RPE cells after subretinal injections, but little or no expression after intravitreal injections13.
For effective gene delivery to RPE, viral promoters should be also considered. Viral promoters, such as cytomegalovirus (CMV) promoter and chimeric chicken beta-actin/CMV enhancer promoter are strong and ubiquitous promoters, thus, they are used in majority of gene therapy studies. In RPE, the CMV promoter is estimated to express ten-fold as much protein compared with the RPE65 promoter14. Of importance, the optimal combination of the promoter and viral vectors including serotype should be achieved for better results as demonstrated by superior expression of RPE65 when the human RPE65 promoter are packaged in serotype rAAV2/4 than rAAV2/215. Focusing on gene delivery to RPE cells, RPE-specific promoters such as the RPE65 or the VMD2 promoter reduces the off-target safety issues16.
AMD is a complex disease that has two phases with a degenerative phase referred to as dry AMD and a phase characterized by choroidal neovascularization referred to as wet AMD. Also, AMD is a disease in which numerous genetic susceptibilities including complement factor H (CFH), complement C3, complement factor I (CFI), Age-related maculopathy susceptibility 2 (ARMS2) and ApoE are combined with environmental risk factors17. Due to the complex nature of genetic risk factors in AMD, there is no single candidate gene for its gene therapy. Thus, the current gene therapy in AMD is focused on gene transfer to express anti-angiogenic proteins targeting choroidal neovascularization in wet AMD18. There is no candidate gene yet for treating dry AMD with a gene therapy. Recently, we suggested that intracellular amyloid beta accumulation could be related to dry AMD features in transgenic mice19. In addition, subretinally injected amyloid beta was taken up by RPE in mice (data not published). We can cleave intracellular amyloid beta with viral protease20. Thus, we have further studied the gene therapy in dry AMD with potential targets.
In conclusion, gene therapy in ocular disease is more promising than in any other organ. In addition, subretinal injection is a fundamental technique for gene therapy in retinal diseases, especially in relation to photoreceptor cells and RPE. Thus, we hope that this technique becomes more widely used and can contribute to the advancement of gene therapy not only in monogenic inherited retinopathy but also in age-related macular degeneration.

In ophthalmology, gene therapy has emerged as the treatment modality in monogenic inherited retinopathies. There are inherited retinopathies associated with genes in retinal pigment epithelium (RPE) including Leber congenital amurosis1,2, retinitis pigmentosa3, and choroideremia4. The research field of gene therapy is expanding in both preclinical studies and clinical trials using viral vectors such as adeno-associated virus (AAV), lentivirus (LV) and adenovirus (Ad)5. Different viral vectors have different tropism in the retina. For a safe and effective gene therapy, viral vectors should be carefully selected according to target cells and target genes.
The route of gene delivery is also important for effective gene delivery to target cells, thus, it should be carefully chosen as well. The two most common methods for intraocular delivery of viral vectors are subretinal injection and intravitreal injection6. The latter, intravitreal injection, has been widely used for drug delivery to treat choroidal neovascularization in wet age-related macular degeneration (AMD) and macular edema in diabetic retinopathy7. Intravitreal route provides exposure of viral vectors to vitreous and inner&#160;retina, but the diffusion of the vectors to outer retina is limited. On the other hand, the subretinal route provides direct delivery of viral vectors to the potential space between retina and RPE, inducing a localized bleb. Therefore, subretinal injection is currently considered a more efficient route for targeting photoreceptor cells and RPE. In terms of surgical approach, pars plana is chosen as a safe area for intravitreal injection to avoid retinal damage in human patients. By simply modifying this approach to mice, we could inject viral vectors subretinally or intravireally via limbal approach.
In this video article, we demonstrate an easy and convenient method of subretinal injection of viral vectors into mice RPE. After single puncture at posterior to limbus with a 30 G 1/2 needle, a 33 G blunt needle equipped microliter syringe is inserted into the subretinal space via the limbal puncture site. The viral vectors of 1.5 - 2 &#181;l volume are injected to the potential space between retina and RPE inducing subretinal blebs. This procedure can be performed under direct visualization using surgical microscope. Repeated practice will guarantee replicable results even without direct visualization of the bleb formation. This will help the researchers to perform accurate and timesaving experiments for subretinal gene delivery in mice RPE.

<table><tbody><tr><td>TWEEZERS DUMONT #5 11cm DUMOSTAR 0.1 x 0.06 mm TIPS</td><td>WPI</td><td>500233</td><td></td></tr><tr><td>VANNAS Scissors S/S, 105 mm</td><td>WPI</td><td>555583S</td><td></td></tr><tr><td>33 G Blunt needle</td><td>WPI</td><td>NF33BL-2</td><td></td></tr><tr><td>NanoFil Syringe, 10 microliter&amp;nbsp;</td><td>WPI</td><td>NANOFIL</td><td></td></tr><tr><td>RPE-KIT</td><td>WPI</td><td>RPE-KIT</td><td>For easy one hand injection</td></tr><tr><td>30 G x 1/2 (0.3 mm x 13 mm) BD PrecisionGlide&lt;sup&gt;TM&lt;/sup&gt; Needle</td><td>BD</td><td>305107</td><td>Initial puncture for subretinal injection</td></tr><tr><td>Microscope Cover Glasses (No. 1 3 mm diameter)</td><td>Warner Instruments</td><td>64-0720&amp;nbsp; (CS-3R)</td><td></td></tr><tr><td>Leica operating microscope</td><td>Leica</td><td>LM M80</td><td></td></tr><tr><td>Fluoresecein microscope</td><td>Nikon</td><td>Eclipse 80i</td><td></td></tr><tr><td>Lentivirus</td><td>Thermo scientific</td><td>TMO.LV-Ctr</td><td>Used to dilute vectors</td></tr><tr><td>PBS</td><td>Gibco</td><td>10010-015</td><td>Used to dilute vectors</td></tr><tr><td>Troperin (Phenylephrin 0.5% - Tropicamide 0.5%)</td><td>Hanmi</td><td></td><td>For dilation</td></tr><tr><td>Proparacaine Hydrochloride Ophthalmic Solution USP, 0.5% (Sterile)</td><td>Bausch&amp;amp;Lomb</td><td></td><td>For topical anesthesia</td></tr><tr><td>Healon GV OVD</td><td>Abbott Medical Optics Inc.</td><td></td><td></td></tr><tr><td>Zoletil 50 (tiletamine hypochloride and zolazepam hypochloride)</td><td>Virbac</td><td></td><td>For general anesthesia</td></tr><tr><td>Rompun injection (Xylazine HCl)</td><td>Bayer</td><td></td><td>For general anesthesia</td></tr></tbody></table>

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.

To evaluate the efficacy of the subretinal injection on viral gene transduction by this protocol, we used commercially available LV vectors with CMV promoter expressing both GFP and RFP for the indicator. Eyes were enucleated after the appropriate time period according to the research purpose. For the representative results, eyes were enucleated 10 weeks and 20 weeks after subretinal injection. After complete removal of the retina using the method described above, the flat mount of RPE/choroid/sclera complex was evaluated under fluorescence microscope. The representative results were shown in Figure 3A (good example with complete removal of the retina) and Figure 3B (poor example with intact retina). Although the existence of remnant neural retina (especially photoreceptor outer segment) over RPE layer did not interfere to analyze the area of gene delivery in RPE, it blocked the fluorescence of GFP/RFP and inhibited direct visualization of RPE layer for further analyses. Therefore, complete removal of neural retina from the RPE/choroid/sclera complex would provide better results.
<img alt="Figure 3" src="/files/ftp_upload/53030/53030fig3.jpg" /> 
Figure 3.&#160;The Example of RPE/Choroid/Scleral Complex Flat-mount according to the Complete Removal of Retina. Twenty Weeks after Subretinal Injection of Lentivirus with GFP/RFP Gene. (A) Good example of RPE flat mount with complete removal of the retina shows clear and discrete expression of GFP/RFP in the RPE cells. (B) Poor example of RPE flat mount due to no removal of the retina shows the area of gene delivery with RFP signals which is inadequate to analyze the RPE pathology. Magnification: 40X, Scale bar: 500 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53030/53030fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
It is hard to judge the efficacy by the quantitative analysis because signal intensities of GFP/RFP are variable in the RPE cells of the injected area. In addition, the area of transduction is limited to the area of bleb formation after injection. Thus, it would be more reliable to judge the efficacy by the presence of GFP/RFP expression according to the viral titer per same injected volume (i.e. 2 &#181;l). This helps to adjust appropriate viral titer for the experiment.
The representative results of RPE/choroid/scleral complex flat mount 10 weeks after subretinal injection were shown in various magnification in Figure 4 (40X) and Figure 5 (400X). The images with low magnification are enough to evaluate the area of GFP/RFP expression. On the other hand, the images with high magnification are necessary for other RPE pathology regarding to the researchers&#8217; specific purposes. These images with various magnifications help to understand the viral expression pattern in RPE (Figure 4 and 5). We have also checked the expression of GFP/RFP up to 36 weeks post injection. The expression of GFP/RFP was still observed after 36 weeks post injection (data not shown).
<img alt="Figure 4" src="/files/ftp_upload/53030/53030fig4.jpg" /> 
Figure 4.&#160;The Example of RPE/Choroid/Scleral Complex Flat-mount 10 Weeks after Subretinal Injection of Lentivirus with GFP/RFP Gene (Magnification: 40X).&#160;(A) green channel for GFP (B) red channel for RFP. Scale bar: 500 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53030/53030fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/53030/53030fig5.jpg" /> 
Figure 5.&#160;The Example of RPE/Choroid/Scleral Complex Flat-mount 10 Weeks after Subretinal Injection of Lentivirus with GFP/RFP Gene (Magnification: 400X).&#160;(A) green channel for GFP (B) red channel for RFP. Scale bar: 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53030/53030fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium at JoVE.com

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

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 ...

limbal-approach-subretinal-injection-viral-vectors-for-gene-therapy

Nanyang Technological University

Research

JoVE Journal

Neuroscience

21.0K Views.  Seoul National University College of Medicine. 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.

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

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.2 These include RPE65 gene replacement therapy in patients with Leber's congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt's disease.3-4 Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.5-8 In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue.

This surgical technique illustrates the injection of gene therapy vectors and stem cells into the subretinal space of the mouse eye.

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy ...

Medicine

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment epithelium (RPE) cells. RPE provide critical support for photoreceptors and death or dysfunction of RPE cells is characteristic of age-related macular degeneration (AMD), the leading cause of permanent vision loss in people age 55 and older. While no cure for AMD has been identified, implantation of healthy RPE in diseased eyes may prove to be an effective treatment, and large numbers of RPE cells can be readily generated from pluripotent stem cells. Several interesting questions regarding the safety and efficacy of RPE cell delivery can still be examined in animal models, and well-accepted protocols used to inject RPE have been developed. The technique described here has been used by multiple groups in various studies and involves first creating a hole in the eye with a sharp needle. Then a syringe with a blunt needle loaded with cells is inserted through the hole and passed through the vitreous until it gently touches the RPE. Using this injection method, which is relatively simple and requires minimal equipment, we achieve consistent and efficient integration of stem cell-derived RPE cells in between the host RPE that prevents significant amount of photoreceptor degeneration in animal models. While not part of the actual protocol, we also describe how to determine the extent of the trauma induced by the injection, and how to verify that the cells were injected into the subretinal space using in vivo imaging modalities. Finally, the use of this protocol is not limited to RPE cells; it may be used to inject any compound or cell into the subretinal space.

Here we present a community accepted protocol in multimedia format for subretinally injecting a bolus of RPE cells in rats and mice. This approach can be used for determining rescue potentials, safety profiles, and survival capacities of grafted RPE cells upon implantation in animal models of retinal degeneration.

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment ...

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to the interphotoreceptor/subretinal compartment that has direct exposure to photoreceptors and the retinal pigment epithelium (RPE). Subretinal injections of plasminogen as well as recent preclinical and clinical trials have demonstrated safety and/or efficacy of delivering viral vectors and stem cells to individuals with advanced retinal disease. Mouse models of retinal disease, particularly hereditary retinal dystrophies, are essential for testing these therapies. The most common injection procedure in rodents is to use small transcorneal or transcleral incisions with an anterior approach to the retina. With this approach, the injection needle penetrates the neurosensory retina disrupting the underlying RPE and on insertion can easily nick the lens, causing lens opacification and impairment of noninvasive imaging. Accessing the subretinal space via a transcleral, posterior approach avoids these problems: the needle crosses the sclera approximately 0.5 mm from the optic nerve, without retinal penetration and avoids disrupting the vitreous. Collateral damage is limited to that associated with the focal sclerotomy and the effects of a transient, serous retinal detachment. The simplicity of the method minimizes ocular injury, ensures rapid retinal reattachment and recovery, and has a low failure rate. The minimal damage to the retina and RPE allows for clear assessment of the efficacy and direct effects of the therapeutic agents themselves. This manuscript describes a novel subretinal injection technique that can be used to target viral vectors, pharmacological agents, stem cells or induced pluripotent stem (iPS) cells to the subretinal space in mice with high efficacy, minimal damage, and fast recovery.

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Subretinal injections are the most common technique for delivering large therapeutic agents such as proteins and viral vectors to photoreceptors and the retinal pigment epithelium. An alternative method in mice that successfully targets the subretinal space with minimal collateral damage and fast recovery times is described here.

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to ...

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is ...

Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the subretinal space, and to evaluate toxicity and efficacy in vivo. HR-SD-OCT uses near infrared light (800-880 nm) and has optics specifically designed for the unique optics of the mouse eye with sub-2-micron axial resolution. Transgenic mouse models of outer retinal (photoreceptor) degeneration and controls were imaged to assess the disease progression. Pulled glass microneedles were used to deliver sub retinal injections of adeno-associated virus (AAV) or nanoparticles (NP) via a trans-scleral and trans-choroidal approach. Careful positioning of the needle into the subretinal space was required prior to a calibrated pressure injection, which delivers fluid into the sub retinal space. Real time subretinal surgery was conducted on our retinal imaging system (RIS). HR-SD-OCT demonstrated progressive uniform retinal degeneration due to expression of a toxic mutant human mutant rhodopsin (P347S) (RHOP347S) transgene in mice. HR-SD-OCT allows rigorous quantification of all the retinal layers. Outer nuclear layer (ONL) thickness and photoreceptor outer segment length (OSL) measurements correlate with photoreceptor vitality, degeneration, or rescue. The RIS delivery system allows real-time visualization of subretinal injections in neonatal (~P10-14) or adult mice, and HR-SD-OCT immediately determines success of delivery and maps areal extent. HR-SD-OCT is a powerful tool that can evaluate the success of subretinal surgery in mice, in addition to measuring vitality of photoreceptors in vivo. HR-SD-OCT can also be used to identify uniform animal cohorts to evaluate the extent of retinal degeneration, toxicity, and therapeutic rescue in preclinical gene therapy research studies.

Here we demonstrate a novel approach to using high resolution spectral-domain optical coherence tomography (HR-SD-OCT) to assist delivery of gene therapy agents into the subretinal space, assess its areal coverage, and characterize photoreceptor vitality.

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the ...

Subretinal Transplantation of Human Embryonic Stem Cell-Derived Retinal Tissue in a Feline Large Animal Model

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and Leber Congenital Amaurosis (LCA) cause progressive and debilitating vision loss. There is an unmet need for therapies that can restore vision once photoreceptors have been lost. Transplantation of human pluripotent stem cell (hPSC)-derived retinal tissue (organoids) into the subretinal space of an eye with advanced RD brings retinal tissue sheets with thousands of healthy mutation-free photoreceptors and has a potential to treat most/all blinding diseases associated with photoreceptor degeneration with one approved protocol. Transplantation of fetal retinal tissue into the subretinal space of animal models and people with advanced RD has been developed successfully but cannot be used as a routine therapy due to ethical concerns and limited tissue supply. Large eye inherited retinal degeneration (IRD) animal models are valuable for developing vision restoration therapies utilizing advanced surgical approaches to transplant retinal cells/tissue into the subretinal space. The similarities in globe size, and photoreceptor distribution (e.g., presence of macula-like region area centralis) and availability of IRD models closely recapitulating human IRD would facilitate rapid translation of a promising therapy to the clinic. Presented here is a surgical technique of transplanting hPSC-derived retinal tissue into the subretinal space of a large animal model allowing assessment of this promising approach in animal models.

Presented here is a surgical technique for transplanting human pluripotent stem cell (hPSC)-derived retinal tissue into the subretinal space of a large animal model.

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and ...

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 ...

Intravitreal Injections in the Ovine Eye

There are several methods for the delivery of therapeutic agents to the retina, including intravitreal (IVT), subretinal, suprachoroidal, periocular, or topical administration. IVT drug delivery involves an injection into the vitreous humor of the eye, a gelatinous substance that fills the posterior chamber of the eye and maintains the shape of the eye globe. Although the IVT route is less specifically targeted than subretinal delivery, it is much less invasive and is widely used in clinical settings for a range of ocular diseases. 
We previously demonstrated the efficacy of intravitreal delivery of an adeno-associated virus (AAV)-mediated gene therapy product (AAV9.CLN5) in sheep with a naturally occurring CLN5 form of neuronal ceroid lipofuscinosis (NCL). Affected sheep received IVT gene therapy in one eye, with the other untreated eye serving as an internal control. Retinal structure and function were maintained in the treated eye up to 15 months after treatment, while the untreated eye displayed progressively declining function and severe atrophy during postmortem examination. Based on the sheep studies, the CLN5 gene therapy product was cleared as a candidate investigational new drug (IND) by the United States Food and Drug Administration in September 2021. This paper details the surgical protocol for IVT delivery of a therapeutic viral vector to the ovine eye.

Intravitreal injections were performed in the sheep eye with the aim of delivering viral-mediated gene therapy to the retina.

There are several methods for the delivery of therapeutic agents to the retina, including intravitreal (IVT), subretinal, suprachoroidal, periocular, or ...

Transpupillary-Guided Trans-Scleral Transplantation of Subretinal Grafts in a Retinal Degeneration Mouse Model

Transplantation of photoreceptor cells and retinal pigment&#160;epithelial (RPE) cells provide a potential therapy for retinal degeneration diseases. Subretinal transplantation of therapeutic donor cells into mouse recipients is challenging due to the limited surgical space allowed by the small volume of the mouse eye. We developed a trans-scleral surgical transplantation platform with direct transpupillary vision guidance to facilitate the subretinal delivery of exogenous cells in mouse recipients. The platform was tested using retinal cell suspensions and three-dimensional retinal sheets collected from rod-rich Rho::EGFP mice and cone-rich OPN1LW-EGFP;NRL-/- mice, respectively. Live/dead assay showed low cell mortality for both forms of donor cells. Retinal grafts were successfully delivered into the subretinal space of a mouse model of retinal degeneration, Rd1/NS, with minimum surgical complications as detected by multimodal confocal scanning laser ophthalmoscope (cSLO) imaging. Two months post-transplantation, histological staining demonstrated evidence of advanced maturation of the retinal grafts into &#39;adult&#39; rods and cones (by robust Rho::EGFP, S-opsin, and OPN1LW:EGFP expression, respectively) in the subretinal space. Here, we provide a surgical platform that can enable highly accurate subretinal delivery with a low rate of complications in mouse recipients. This technique offers precision and relative ease of skill acquisition. Furthermore, the technique could be used not only for studies of subretinal cell transplantation but also for other intraocular therapeutic studies including gene therapies.

This protocol presents a transpupillary vision-guided trans-scleral approach to safely and precisely deliver subretinal cellular grafts, with a low rate of surgical complications, in mouse recipients with or without retinal degeneration.

Transplantation of photoreceptor cells and retinal pigment&#160;epithelial (RPE) cells provide a potential therapy for retinal degeneration diseases. ...

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

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