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

Bu çalışma Seul Ulusal Üniversitesi Araştırma Grant (800-20140542) tarafından desteklenen, NTG / EBTB Pioneer Araştırma Programı (2012-0009544) ve NRF / EBTB Bio-Sinyal Analizi Teknoloji İnovasyon Programı (2009-0090895), ve NRF / EBTB Hibe (2015M3A9E6028949).

Hayvanlar üzerinde deneyler tüm Oftalmik ve Vizyon Araştırma Hayvanların Kullanım Vizyon ve Oftalmoloji Tablosunda Araştırma Derneği göre yapılan ve kurallar ve yönetmelikler Seul Ulusal Üniversitesi Kurumsal Hayvan Bakım ve Kullanım Komitesi ve Seul Ulusal öngördüğü edildi Üniversite Hastanesi Biyogüvenlik Komitesi. 1. Enjeksiyon Kit ve Viral Vektörler hazırlanması <ol><li> Etilen oksit gazı ile sterilize edilmiş bir 33 G, kör bir iğne ile donatılmış mikrolitre şırınga hazırlayın. Mikro tüp (yani, 1 x 10 6 TU / ml) &#39;de yeterli bir titre PBS içinde viral vektörleri ile seyreltilir. Şırınga herhangi ölü boşluğu kaldırmak için enjektörü viral vektörler ile birkaç kez yıkayın. </li></ol> Viral vektörler 2. Subretinal Enjeksiyon <ol><li> Tiletami karışımının intraperitoneal enjeksiyonu ile - (8 haftalık, yani, 6), yetişkin fareler anesteziKD ve zolazepam (1: 1, 2.25 mg / kg vücut ağırlığı) ve ksilazin hidroklorür (0.7 mg / kg vücut ağırlığı) ya da alternatif olarak uygun anestezi rejimi. </li><li> Fenilefrin% 0.5 göz damlası ile öğrenciler dilate ve% 0.5 tropikamid. </li><li> Viral vektörlerin 2 ul - 1.5 ile yüklenerek mikrolitre şırınga hazırlayın. </li><li> Göz kapağı açın ve uygun enjeksiyon için ekvator ortaya çıkarmak ve işletim mikroskop altında odaklanmak göz çıkıntı. Enjeksiyon sırasında oluşabilecek iğne enjeksiyonu veya deplasman bitirme kadar çıkıntı göz pozisyonunu koruyun. Sıkıca göz küresi tutun, yörünge jant dışında parmaklarını yerleştirin. </li><li> Kornea yüzeyine oftalmik viskoelastik bir çözelti damla uygulanır. </li><li> Retina görselleştirmek için kornea üstünde küçük bir yuvarlak kapak slayt yerleştirin. </li><li> Daha fazla subretinal enjeksiyon için steril 30 G 1/2 iğne kullanılarak limbusa hafif posterior küçük bir delik delmek. Fo delik aşağı olunr sağ göz ve rahatlık için sol göz için üstün. NOT: delik zamansal ya da nazal yapılır ise, enjeksiyondan sonra göz kapağında kapsamına zor. İlk ponksiyon hafifçe yapılan limbusun boyunca çalışacak ve kolayca tanınabilir limbal damarları önlemek için limbusa posteriorunda edilmelidir. İlk ponksiyon yapılırken iğne ile lensi çarpmamak için dikkatli olun. Lens ponksiyon önlemek için iğnenin tüm konik takmayın. </li><li> Önceden delinmiş delikten mikrolitre şırınga 33 G künt iğne yerleştirin ve hafif direnç hissedilir zaman noktaya kadar subretinal boşluğa iğne yaklaşım. NOT: subretinal enjeksiyon için, iğnenin en iyi yaklaşım açısı iris düzleminin karşı yaklaşık 45 derece, ve künt bir iğne peripapiller alana doğru posterior itti olmalıdır. Noktalı kare subretinal injectio için vitreus boşluğuna genelinde önerilen iğne yolu belirtilenŞekil 1 &#39;de: n. NOT: Onlar çok yumuşak olduğu gibi delici (veya geçen) retina katmanları zaman direnç hissettim olacak. Böylece, hafif direnç ilk duygu iğne zaten RPE dokundu ve iğnenin takılması durdurulması gerektiğini belirtir. İğne potansiyel subretinal alana konulmalıdır çünkü aşırı basınç skleral doku nüfuz için dikkatli olun. İğne aşırı güç tarafından skleral doku deler, bu direnç olmadan göz dışı yörünge alanlarda girer. (Bu adım önemlidir) </li></ol><img alt="Figür 1" src="/files/ftp_upload/53030/53030fig1.jpg" /> Subretinal Enjeksiyon Şekil 1. Şematik. H &amp; E işaretli subretinal enjeksiyon için bir iğne yolunun olan yapıları resmeden fare göz lekeli kesiti (noktalı kare). Büyütme: 40X, Ölçek çubuğu:<html> 500 mikron. <a href="https://www.jove.com/files/ftp_upload/53030/53030fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <ol start="9"><li> İstenmeyen doku hasarını önlemek ve yavaşça iğneyi çekilme titreme olmadan subretinal boşluğa yavaşça viral vektörler (örneğin, 1 x 10 6 TU / ul) enjekte edilir. 2.4&#39;te tarif edildiği gibi enjeksiyon sırasında sıkıca gözün tutun. </li><li> Hiçbir retina kanaması olduğundan emin olmak için, işletim mikroskop altında enjekte edildikten sonra subretinal bleb oluşumunu gözlemleyin. </li></ol><img alt="Şekil 2," src="/files/ftp_upload/53030/53030fig2.jpg" /> Şekil 2. Retina Kanama olmadan Subretinal BELB Formasyonu. Işletim mikroskop altında subretinal enjeksiyondan sonra subretinal BELB mikroskopik görünümü retinal kanama olmadan başarılı bleb oluşumu göstermektedir.m / files / ftp_upload / 53030 / 53030fig2large.jpg &quot;target =&quot; _ blank &quot;&gt; bu rakamın daha büyük bir versiyonunu görmek için lütfen buraya tıklayınız. <ol start="11"><li> Yavaşça kendini sızdırmazlık için enjeksiyon yerinde kapsayacak şekilde göz kapaklarını kapatın. Ev kafesine fareler dönün ve değerlendirme kadar canlı tutmak. NOT: Araştırmacılar prosedürlere alışması sonra, aşağıdaki adımları atlayarak hızlı prosedürlere tekrarlanabilir sonuçlar elde (2.5, 6, ve 10) olabilir </li></ol> Retina Pigment Epitel Gen Teslimat Etkinliğinin Değerlendirilmesi 3. <ol start="1"><li> CO2 odası içinde fareler kurban. Makas kullanılarak gözleri enükleasyon. </li><li> Birkaç gün için en az 1 saat boyunca 4 ° C &#39;de% 4 paraformaldehit solüsyonunda gözleri düzeltildi. </li><li> Ince forseps ile kornea tut ve mikro-makas ile kornea delinme. Limbusun boyunca makaslama sonra kornea çıkarın. Forseps ile lensi çıkarın. </li><li> M siliyer cisim Trimretina dekolmanı izin ICRO-makas. Yavaşça RPE / koroid / sklera karmaşık bütün retinanın çekin. Düz montaj için 4 ila 8 yaprakları yapmak için optik sinir birkaç kez yakın merkeze çevresinden radyal RPE / koroid / sklera kompleksi kesin. </li><li> Aşağı RPE tarafı yapmak ve kas, konjonktiva ve optik sinir dahil skleral tarafında kalan tüm dokuları kesmek için RPE / koroid / sklera kompleksi çevirin. Kalan doku, karmaşık düzensiz hale getirir, çünkü bu adım, düz RPE / koroid / sklera kompleksi yapmak önemlidir. </li><li> (Ayrıntılı yöntemleri için referanslara bakın, isteğe bağlı) özel araştırma amaçlı birincil ve ikincil antikor ile kompleks inkübe edin. Cam slayt RPE / koroid / sklera kompleksleri Düz montaj ve emici bir sünger ile PBS emer. </li><li> Montaj çözümü 30 ul ekleyin ve kapağını kaydırarak yerleştirin. Floresein mikroskop altında vektör teslim etkinliği için örnek gözlemleyin. Örnekleri tutunDaha fazla gözlem için buzdolabı. </li></ol>

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The authors have nothing to disclose.

Bu video makalede, biz RPE / koroid / sklera düz montaj temsilcisi sonuçları ile detaylı limbal-yaklaşım subretinal enjeksiyon tekniği nitelendirdi. Bu RPE viral vektörlerin retina altı enjeksiyon için kolay ve uygun bir tekniktir. Enjeksiyon sırasında kabarcık oluşumu doğrudan görselleştirme yeni başlayanlar için doğru bir teslimat için önemli bir adımdır. Visualized Experiments 8-10 Journal tanıtılan bazı subretinal enjeksiyon teknikleri vardır. Subretinal alan retina ve RPE aras...

Oftalmolojide, gen terapisi monogenik miras retinopatilerin tedavi yöntemi olarak ortaya çıkmıştır. Leber konjenital amurosis 1,2, retinitis pigmentosa 3 ve Koroideremi 4 olmak üzere retina pigment epiteli (RPE) genlerin ilişkili kalıtsal retinopatiler vardır. gen tedavisi araştırma alanı preklinik çalışmalarda ve adeno virüsü enzimi yüksek yoğunluklu lipoproteinlerde (AAV), lentivirüs (LV) ve adenovirüs (Ad) gibi viral vektörler kullanılarak klinik çalışmalarda hem de genişliyor 5. Farklı viral vektörler retina farklı tropizm ihtiva. Güvenli ve etkili bir gen terapisi için viral vektörler, dikkatli bir şekilde hedef hücreler ve hedef genlerin uygun olarak seçilmelidir. etkili bir gen verici hedef hücrelere gen teslim yolu bu nedenle, dikkatli bir şekilde da seçilmelidir, da önemlidir. viral vektörlerin göz içi teslimat için en yaygın iki yöntemler subret edilirinal enjeksiyon ve intravitreal enjeksiyon 6. İkincisi, intravitreal enjeksiyon, yaygın diyabetik retinopati 7 ıslak yaşa bağlı makula dejenerasyonu (YBMD) ve maküla ödemi koroid neovaskülarizasyon tedavisi için ilaç verilmesi için kullanılır olmuştur. İntravitreal rota vitreus ve iç retina viral vektörlerin pozlama sağlar, ancak dış retinaya vektörlerin difüzyon sınırlıdır. Öte yandan, alt retinal yol lokalize bir kabarcık indükleyici, retina ve RPE arasındaki potansiyel boşluğa viral vektörlerin doğrudan dağıtım sağlar. Bu nedenle, alt retinal enjeksiyon anda fotoreseptör hücreleri ve RPE hedefleme için daha verimli bir yol olarak kabul edilir. Cerrahi yaklaşım açısından, plana insan hastalarda retina hasarı önlemek için intravitreal enjeksiyon için güvenli bir alan olarak seçilmiştir pars. Sadece farelere bu yaklaşımı değiştirerek, biz limbal yaklaşımla subretinally veya intravireally viral vektörlerin enjekte edebilir. Bu videodamakale, biz fareler RPE viral vektörlerin subretinal enjeksiyonu kolay ve uygun bir yöntem ortaya koymaktadır. 30 G 1/2 iğne ile limbusa posterior tekli delme sonra, 33 G iğne künt donanımlı mikrolitre şırınga limbal ponksiyon sitesi üzerinden subretinal boşluğa yerleştirilir. 1.5 viral vektörler - 2 ul hacminde, retina ve RPE subretinal kabarcıkları indükleyici arasındaki potansiyel boşluğa enjekte edilir. Bu prosedür, cerrahi mikroskop kullanılarak doğrudan görüş altında gerçekleştirilebilir. Tekrarlanan uygulama bile kabarcık oluşumu doğrudan görselleştirme olmadan tekrarlanabilir sonuçları garanti edecektir. Bu fareler RPE subretinal gen aktarımı için doğru ve zaman kazandıran deneyler gerçekleştirmek için araştırmacıların yardımcı olacaktır.

<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.

Bu protokol ile viral gen transdüksiyon üzerinde subretinal enjeksiyonunun etkinliğini değerlendirmek için, göstergesi GFP ve RFP hem ifade CMV promotör ile piyasada mevcut LV vektörleri kullanılır. Gözler araştırma amacına göre uygun süre sonra iki parçaya ayrıldı. Temsilcisi sonuçları için gözler 10 hafta ve subretinal enjeksiyondan sonra 20 hafta parçaya ayrıldı. Yukarıda tarif edilen yöntem kullanılarak, retina tamamen uzaklaştırıldıktan sonra, RPE / koroid / sklera kompleksinin düz...

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.

Fareler Retina Pigment Epitel Gen Tedavisi için Viral Vektörler Limbal Yaklaşım-Subretinal Enjeksiyon

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 Görüntüleme.  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.

Fare Göz içine Gen Terapisi Vektörler ve Kök Hücre Subretinal Enjeksiyon

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 ...

Tıp

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.

Sodyum Hyaluronat subretinal Enjeksiyon Kemirgenler Retina Dekolmanı Modeli

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.

Süspansiyon Retina pigment epiteli Hücreleri sunun Kemirgenler Subretinal Enjeksiyonları Sahne

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 Space erişim için bir alternatif ve Onaylı Enjeksiyon Yöntemi<i&gt; yoluyla</i&gt; Bir transkleral Posterior Yaklaşım

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

Nörobilim

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.

Sıçan gözlerinin içine insan retina Pigment epitel hücrelerinin Trans-scleral Subretinal nakli için rafine bir protokol geliştirilmesi

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.

Ultrahigh çözünürlük fare optik koherens tomografi göz içi enjeksiyon Retina gen terapisi araştırma yardımcı olmak için

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.

Bir Kedi Büyük Hayvan Modelinde İnsan Embriyonik Kök Hücre Kaynaklı Retinal Dokunun Subretinal Transplantasyonu

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.

Küçük Hayvan Modellerinde Adeno İlişkili Virüs Vektörlerinin Subkonjonktival Uygulaması

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.

Küçükbaş Gözde İntravitreal Enjeksiyonlar

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.

Retinal Dejenerasyon Fare Modelinde Subretinal Greftlerin Transpupiller Kılavuzluğunda Transskleral Transplantasyonu

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