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

这项研究是由首尔国立大学研究资助（800-20140542）的支持下，先锋研究计划NRF / MEST的（2012-0009544），和NRF / MEST的生物信号分析技术创新计划（2009-0090895），及NRF / MEST的格兰特（2015M3A9E6028949）。

对动物的所有实验均按照研究协会在视觉与眼科声明使用动物的眼科和视觉研究的进行，以及指导方针和条例规定由首尔国立大学机构动物护理和使用委员会和国立首尔大学附属医院生物安全委员会。 1.准备注射套件和病毒载体<ol><li>制备微升注射器配备有33克钝针用环氧乙烷气体消毒。稀释在PBS中的病毒载体用于在微管（ 即，1×10 6 TU /微升）在足够滴度。冲洗注射器数次用病毒载体以除去在注射器任何死角。 </li></ol>病毒载体2.视网膜下注射<ol><li>麻醉成年小鼠（ 即，6 - 8周龄）腹膜内注射tiletami的混合物的NE和唑拉西泮（1:1，2.25毫克/公斤体重）和赛拉嗪盐酸盐（0.7毫克/公斤体重）或另一种合适的麻醉制度。 </li><li>瞳孔扩张与去氧肾上腺素0.5％滴眼剂及托0.5％。 </li><li> 2μl病毒载​​体 - 通过加载1.5准备的注射器微升。 </li><li>打开眼睑和突出眼睛，以暴露赤道注射方便和在手术显微镜下重点关注。保持凸眼睛的位置，直到在完成注射针，或位移可以发生在注射过程。要牢牢占据眼球，将手指眶缘外。 </li><li>申请一滴眼科粘弹性溶液到角膜表面。 </li><li>放置在角膜上以可视化的视网膜的顶部小圆盖滑动。 </li><li>穿刺小孔后略有使用无菌30 ​​G 1/2针为进一步视网膜下注射角膜缘。让孔劣质FOr处的右眼，和优越的用于左眼为方便。 注:如果孔是由在时间或鼻腔，很难涵盖在注射后的眼睑。初始穿刺应略作出后侧到角膜缘，以避免其沿角膜缘运行，并且可以容易地识别该边缘血管。要小心，以避免撞上镜头针，而在初始穿刺。不要插入针头的整个斜面，以避免爆胎的镜头。 </li><li>将微升注射器33克钝针经过前期的穿刺孔，并接近针进入视网膜下腔，直到点，当轻微的感觉到阻力。 注:对于视网膜下注射时，针最好接近角是针对虹膜平面约45度，并且钝针应当向后推向乳头周围区域。虚线方框表示整个玻璃体腔建议针途径视网膜下注射液中的n 图1。 注:不会有阻力觉得当刺穿（或合格）的视网膜层，因为他们是非常柔软。因此，温和阻力的第一感觉指示该针已触到RPE层和所述针的插入应当停止。小心不要穿透巩膜组织过度的压力，因为在针应放置在潜在视网膜下腔。如果针头穿刺由剩余电力的巩膜组织，其进入无阻力眼外轨道的空间。 （这一步是至关重要的） </li></ol><img alt="图1" src="/files/ftp_upload/53030/53030fig1.jpg" /> 在视网膜下注射的图1示意图。H＆E的小鼠眼描绘的结构用针通路为视网膜下注射标记的染色的横截面（虚线正方形）。放大倍数:40X，比例尺:<html> 500微米。 <a href="https://www.jove.com/files/ftp_upload/53030/53030fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="9"><li>轻轻注入病毒载体（ 即，1×10 6 TU /微升）到视网膜下腔无震颤，以避免不必要的组织损伤，并轻轻拔出针头。在2.4中描述的注射过程中牢牢占据眼球。 </li><li>观察注射手术显微镜下后形成的水泡视网膜下，以确保没有视网膜出血。 </li></ol><img alt="图2" src="/files/ftp_upload/53030/53030fig2.jpg" /> 图2.视网膜下Belb没有形成视网膜出血，视网膜下注射手术显微镜下视网膜后的belb微观角度显示没有视网膜出血成功水泡形成。M /文件/ ftp_upload / 53030 / 53030fig2large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <ol start="11"><li>轻轻合上眼睑覆盖注射部位为自密封。返回小鼠家笼永葆直到评价。 注:在研究人员习惯的程序，它们可以通过跳过这些步骤获得快速的程序可重复的结果（2.5，6和10） </li></ol> 3.评估基因传递的视网膜色素上皮细胞的功效<ol start="1"><li>牺牲小鼠中的CO 2室中。用剪刀剜除眼睛。 </li><li>固定在眼睛中，在4℃的4％多聚甲醛溶液至少1小时至数天。 </li><li>抢用细镊子角膜和穿刺微型剪刀角膜。沿缘剪式后取下眼角膜。取出钳镜头。 </li><li>修剪睫状体具有mICRO剪刀使视网膜脱离。轻轻一拉从RPE /脉络膜/巩膜复杂的整个视网膜。切RPE /脉络膜/巩膜复杂的径向从外围向中心靠近视神经几次，以4〜8叶平安装。 </li><li>翻转RPE /脉络膜/巩膜复杂，使RPE的一面朝下，并修剪在巩膜侧的肌肉，包括，结膜和视神经所有剩余的组织。这个步骤是重要的，以一平的RPE /脉络膜/巩膜复杂的，因为任何残留组织使复杂的不均匀。 </li><li>孵育一级和二级抗体复合物为特定的研究目的（可选，请参阅详细方法的引用）。平面安装的RPE /脉络膜/巩膜载玻片上复合物和吸收的PBS用吸水海绵。 </li><li>加入30微升的安装解决方案，然后将盖子滑。观察样品载体递送的荧光显微镜下的功效。保持的样品中的冰箱进一步观察。 </li></ol>

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The authors have nothing to disclose.

在这个视频文章中，我们介绍了缘，方法视网膜下注射技术详细RPE /脉络膜/巩膜平座的代表性成果。这是一个简单而方便的技术为视网膜下注射病毒载体进入RPE。的气泡形成在注射过程中直接​​可视化是准确的交货期为初学者的一个重要步骤。有在Journal引入可视化实验8-10中某些视网膜下注射技术。视网膜下空间是视网膜和RPE之间的潜在空间，因此有两种可能的路由接近视网膜下的空间...

在眼科，基因治疗已成为治疗方式在单基因遗传性视网膜病变。存在与基因在视网膜色素上皮细胞（RPE）包括莱伯先天性amurosis 1,2，色素性视网膜炎3，和脉络膜4相关遗传性视网膜病变。基因治疗的研究领域正在扩大在临床前研究和使用病毒载体的临床试验，例如腺机相关病毒（AAV）， 慢病毒 （LV）和腺病毒 （广告）5。不同的病毒载体具有不同的取向，在视网膜上。一种安全有效的基因治疗，病毒载体应仔细根据靶细胞和靶基因。 基因递送途径也很重要，为有效的基因递送至靶细胞，因此，应该仔细选择为好。用于眼内递送病毒载体的两种最常见的方法是subretINAL注射玻璃体内注射6。后者，玻璃体内注射，已被广泛地用于药物输送到治疗在湿性年龄相关性黄斑变性（AMD）和黄斑水肿脉络膜新生血管形成，糖尿病性视网膜病7。玻璃体内途径提供的病毒载体到玻璃体和视网膜内层的暴露，但向量到外视网膜的扩散受到限制。另一方面，在视网膜下途径提供直接递送病毒载体到视网膜和RPE之间的潜在空间，诱导局部疱。因此，视网膜下注射是目前被认为用于靶向感光细胞和RPE一个更有效的路由。在手术方式上，玻璃体被选为安全区的玻璃体内注射，避免在人类患者视网膜损伤。通过简单地修改这个方法给小鼠，我们可以通过角膜缘视网膜下的方法或intravireally注入病毒载体。 在这个视频本文中，我们证明视网膜下注射病毒载体的容易且方便的方法到小鼠视网膜色素上皮。在后方缘用30 G 1/2单针穿刺后，一个33克钝针配备微升注射器插入通过角膜缘穿刺部位的视网膜下腔。 1.5病毒载体 - 2微升体积被注入到视网膜和RPE诱导视网膜下泡之间的潜在空间。这个过程可以在直视下使用手术显微镜下进行。反复练习将保证复制的结果，即使没有气泡形成的直接可视化。这将有助于研究人员进行精确和节省时间用于实验的小鼠视网膜色素上皮基因传递。

<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.

通过该协议评估视网膜下注射病毒基因转导的功效，我们用巨细胞病毒启动子表达GFP和RFP的指标市售LV载体。根据研究目的合适的时间后，眼睛被摘除。对于代表性的结果，眼睛被剜出10周20周视网膜下注射后。完全除去使用上述方法的视网膜后，RPE /脉络膜/巩膜复杂的平面安装在荧光显微镜下进行了评价。代表性结果示于图3A（与完全除去视网膜的很好的例子）和图3B（?...

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.

病毒载体的基因治疗的小鼠视网膜色素上皮缘方针，视网膜下注射

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

Research

JoVE Journal

Neuroscience

21.0K 次观看.  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.

视频: 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.

基因治疗的载体和干细胞视网膜下注射到老鼠的眼睛

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 ...

科研

医学

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.

视网膜脱离模型鼠害由透明质酸钠的视网膜下注射

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.

视网膜下进行注射的鼠害提供视网膜色素上皮细胞悬浮

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.

用于访问视网膜下腔的另类和验证注射法<i&gt;通过</i&gt;一经巩膜后路

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

神经科学

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.

人视网膜色素上皮细胞向大鼠眼巩膜视网膜移植精制方案的研制

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.

超高分辨率小鼠光学相干层析成像辅助眼内注射在视网膜基因治疗中的应用

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.

猫大型动物模型中人类胚胎干细胞来源的视网膜组织的视网膜下移植

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.

小动物模型中腺相关病毒载体的结膜下给药

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.

绵羊眼玻璃体内注射

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.

经瞳孔引导下视网膜下移植物的视网膜下移植物经巩膜移植视网膜变性小鼠模型

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