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

본 연구는 서울 대학교 연구 그랜트 (800-20140542)에 의해 지원되었다, NRF / 교육 과학 기술부의 개척자 연구 프로그램 (2012-0009544)과 NRF / 교육 과학 기술부의 바이오 신호 분석 기술 혁신 프로그램 (2009-0090895) 과 NRF / 교육 과학 기술부의 그랜트 (2015M3A9E6028949).

동물에 대한 모든 실험은 안과 및 비전 연구에서 동물의 사용에 대한 비전 및 안과 문에 연구를위한 협회에 따라 수행하고, 지침과 규정은 서울 대학교 기관 동물 관리 및 사용위원회와 서울 대학교가 정한했다 대학 병원 바이오 안전성위원회. 1. 주입 키트 및 바이러스 성 벡터를 준비 <ol><li> 에틸렌 옥사이드 가스를 사용하여 멸균 33 G 무딘 바늘을 갖춘 마이크로 리터 주사기를 준비한다. 마이크로 튜브 (즉, 1 × 6 TU / μL)의 적절한 역가에 대한 PBS에서 바이러스 벡터를 희석. 주사기 임의 사강을 제거하는 주사기를 바이러스 벡터로 수회 세척. </li></ol> 바이러스 성 벡터의 2. 망막 주입 <ol><li> tiletami의 혼합물의 복강 내 주사 - (8 주 오래 즉, 6) 성인 쥐를 마취NE 및 zolazepam (1 : 1, 2.25 ㎎ / ㎏ 체중)과 자일 라진 하이드로 클로라이드 (0.7 ㎎ / ㎏ 체중) 또는 다른 적절한 마취 정권. </li><li> 페닐에 0.5 %의 점안제와 동공 팽창 및 0.5 % tropicamide. </li><li> 바이러스 벡터의 2 μL - 1.5로드하여 마이크로 리터 주사기를 준비합니다. </li><li> 눈꺼풀을 열고 편리 주입 적도 노출 및 운영 현미경에 초점을 눈 돌출. 주입 동안 발생할 수있는 바늘의 주입, 또는 변위 마무리까지 돌출 눈 위치를 유지한다. 단단히 안구를 유지하기 위해, 궤도 림 외부에서 손가락을 놓습니다. </li><li> 각막 표면 안과 점탄성 용액 한 방울을 적용한다. </li><li> 망막을 시각화하는 각막의 상단에 작은 원형 커버 슬라이드를 놓습니다. </li><li> 추가적인 망막 주 사용 멸균 1/2 30 G 바늘을 사용 윤부에 약간 후방에 작은 구멍이 천공. FO 구멍이 열등 확인R 우안과 편의상 좌안 우수한. 주 : 구멍 시간적 또는 비강에서 이루어진다면, 그것은 주입 후 눈꺼풀에 의해 커버되기 어렵다. 초기 구멍은 약간 만든 윤부를 따라 실행하고 쉽게 인식 할 수 윤부 혈관을 피하기 위해 윤부에 후부해야합니다. 초기 구멍을하는 동안 바늘로 렌즈를 타격 않도록주의하십시오. 렌즈 구멍을 피하기 위해 바늘의 전체 경사를 삽입하지 마십시오. </li><li> 사전 천공 구멍을 통해 마이크로 리터 주사기의 33 G 무딘 바늘을 놓고 가벼운 저항이 느껴지는 경우 지점까지 망막 하 공간에 바늘을 접근. 참고 : 망막 주사를 들어, 바늘의 가장 좋은 방법 각도는 조리개면에 대해 약 45도이며, 무딘 바늘은 유두 주위 지역을 향해 후방으로 밀어해야합니다. 점선 사각형은 망막 injectio의 유리 체강에서 제안 된 바늘의 경로를 표시그림 1에서 N. 참고 : 그들은 매우 부드럽고으로 관통 (또는 전달) 망막 층 경우에는 저항이 느끼지있을 것이다. 따라서, 온화한 저항 제 느낌 바늘 이미 RPE 층을 터치하고 바늘의 삽입이 중단되어야 함을 나타낸다. 바늘이 잠재적 인 망막 공간에 배치해야하기 때문에 과도한 압력과 공막 조직에 침투하지 않도록주의하십시오. 바늘이 과도한 전력에 의해 공막 조직을 천공하면, 저항없이 안구 궤도 공간으로 들어간다. (이 단계는 매우 중요하다) </li></ol><img alt="그림 1" src="/files/ftp_upload/53030/53030fig1.jpg" /> 망막 사출 그림 1. 회로도. H &amp; E 표시 망막 주사 바늘 경로와 구조를 묘사 마우스 눈의 스테인드 단면 (점선 사각형). 배율 : 40X, 스케일 바 :<html> 500 μm의. <a href="https://www.jove.com/files/ftp_upload/53030/53030fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <ol start="9"><li> 불필요한 조직 손상을 방지하고 조심스럽게 바늘을 철회 떨림없이 망막 하 공간에 부드럽게 바이러스 벡터 (즉, 1 × 6 TU / μL)를 주입한다. 2.4에 설명 된대로 주입하는 동안 단단히 안구를 잡습니다. </li><li> 더 망막 출혈이없는 것을 확인하기 위해 운영 현미경으로 주사 후 망막 여과포의 형성을 관찰한다. </li></ol><img alt="그림 2" src="/files/ftp_upload/53030/53030fig2.jpg" /> 그림 2. 망막 출혈없이 망막 Belb 형성. 운영 현미경으로 망막 주사 후 망막 belb의 현미경보기 망막 출혈없이 성공적으로 여과포 형성을 보여줍니다.M / 파일 / ftp_upload / 53030 / 53030fig2large.jpg &quot;대상 =&quot;_ 빈 &quot;&gt;이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오. <ol start="11"><li> 부드럽게 자체 밀봉 용 주사 부위를 덮도록 눈꺼풀을 닫을. 홈 케이지에 마우스를 반환하고, 평가까지 살아 유지. 참고 : 연구자가 절차에 익숙해 후, 다음 단계 건너 뛰어 신속한 절차에 반복 가능한 결과를 얻을 수 (2.5, 6, 10) 수 </li></ol> 망막 색소 상피의 유전자 전달의 효능 3. 평가 <ol start="1"><li> CO 2 챔버에서 쥐를 희생. 가위를 사용하여 눈을 명백히하다. </li><li> 며칠에 최소 1 시간 동안 4 ℃에서 4 % 파라 포름 알데히드 용액에서 쳐다. </li><li> 미세 집게로 각막을 잡고 마이크로 가위로 각막에 구멍. 윤부를 따라 가위질 한 후 각막을 제거합니다. 집게로 렌즈를 제거합니다. </li><li> M와 모양체 트림망막 박리를 허용 icro 가위. 조심스럽게 RPE / 맥락막 / 공막 단지에서 전체 망막을 잡아 당깁니다. 평면 실장 용 4~8 잎 있도록 시신경 수회 가까운 중심 둘레로부터 반경 RPE / 맥락막 / 공막 복합체를 잘라. </li><li> 아래 망막 색소 상피면을 만들고, 근육, 결막 및 시신경을 포함하는 공막 측에 남아있는 모든 조직을 손질 할 수있는 RPE / 맥락막 / 공막 복잡한 플립. 나머지 조직은 복잡한 요철 구조를 만들기 때문에이 단계는 플랫 RPE / 맥락막 / 공막 복합체를 확인하는 것이 중요하다. </li><li> (자세한 방법에 대한 내용을 참조, 선택 사항) 특정 연구 목적으로 기본 및 보조 항체 복합체를 품어. 유리 슬라이드에 RPE / 맥락막 / 공막 단지를 플랫 마운트 흡수성 스폰지로 PBS를 흡수한다. </li><li> 장착 솔루션의 30 μl를 추가하고 커버 슬라이드를 배치합니다. 형광 현미경 벡터 전달의 효능에 대한 샘플을 관찰한다. 에 샘플을 유지더 관찰 냉장고. </li></ol>

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The authors have nothing to disclose.

이 비디오 문서에서는, 우리는 RPE / 맥락막 / 공막 평면 마운트의 대표적인 결과를 상세하게 윤부-접근 망막 주입 기술을 설명했다. 이것은 망막 색소 상피에 바이러스 벡터의 망막 주입을위한 쉽고 편리한 기술이다. 주입시 여과포 형성의 직접 시각화는 초보자를위한 정확한 전달을위한 중요한 단계입니다. 시각 실험 8-10의 저널에 소개 된 일부 망막 사출 기술이있다. 망막 하 공간이 망?...

안과에서, 유전자 치료는 단일 유전자 상속 망막증의 치료법으로서 등장했다. Leber 씨 선천성 amurosis 1,2, 색소 성 망막염 (3), (4)를 포함 choroideremia 망막 색소 상피 (RPE) 유전자와 연관된 상속 망막 병증이있다. 유전자 치료의 연구 분야는 전임상 연구 및 아데노 -associated 바이러스 (AAV), 렌티 바이러스 (LV) 및 아데노 바이러스 (AD)와 같은 바이러스 벡터를 사용하여 임상 시험 모두에서 확대되고있다 (5). 다른 바이러스 성 벡터는 망막에 다른 친 화성이있다. 안전하고 효과적인 유전자 치료의 경우, 바이러스 벡터 신중 표적 세포와 표적 유전자에 따라 선택되어야한다. 효율적인 유전자 전달이 표적 세포에 대한 유전자 전달의 경로는 따라서 신중뿐만 선택되어야한다, 또한 중요하다. 바이러스 벡터의 인공 전달을위한 가장 일반적인 두 가지 방법이 subret된다원고 판 주입과 유리체 강내 주입 6. 후자 주입술 널리 당뇨 망막 7 습식 노인성 황반변 성 (AMD) 및 황반부 종의 맥락막 혈관 신생을 치료하는 약물 전달을 위해 사용되었다. 유리체 강내 경로 및 내측 망막 바이러스 벡터의 노광을 제공하지만, 외측 망막 벡터의 확산이 제한된다. 한편, 망막 경로 지역화 여과포를 유도, 망막 및 RPE 간의 잠재적 바이러스 벡터 공간으로의 직접 전달을 제공한다. 따라서, 망막 하 주사 현재 시각 세포 및 RPE를 타겟팅보다 효율적인 경로로 간주된다. 수술 방법의 측면에서, 인간 환자 유리체 망막 손상을 방지하기 위해 주입술 안전한 영역으로서 선택된다 갈 거예요. 단순히 쥐에이 방법을 수정하여, 우리는 윤부 접근 방식을 통해 subretinally 또는 intravireally 바이러스 벡터를 주입 할 수있다. 이 비디오에서기사, 우리는 쥐의 망막 색소 상피에 바이러스 벡터의 망막 주사의 쉽고 편리한 방법을 보여줍니다. 30 G 1/2 바늘 윤부에 후방에서 단일 천자 후, 33 G 무딘 바늘 장착 된 마이크로 리터 주사기는 윤부 천자 사이트를 통해 망막 하 공간에 삽입됩니다. 1.5의 바이러스 성 벡터 - 2 μL 볼륨은 망막과 망막 색소 상피가 망막 소포를 유도 사이의 잠재적 인 공간으로 주입된다. 이 절차는 수술 현미경을 사용하는 직접적인 시각화 하에서 수행 될 수있다. 반복 연습도 여과포 형성의 직접 시각화없이 복제 결과를 보장합니다. 이 쥐의 망막 색소 상피에서 망막 유전자 전달에 대한 정확하고 시간을 절약 실험을 수행하는 연구자 도움이 될 것입니다.

<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.

이 프로토콜에 의해 바이러스 성 유전자 전달에 망막 주사의 효능을 평가하기 위해, 우리는 인디케이터 GFP 및 RFP를 모두 표현하는 CMV 프로모터와 시판 LV 벡터를 사용했다. 눈은 연구 목적에 따라 적절한 시간 후에 적출 하였다. 대표적인 결과를 들어, 눈을 10 주에서 망막 주사 후 20 주 적출 하였다. 전술 한 방법을 사용하여 망막의 완전한 제거 후, ​​RPE / 맥락막 / 공막 복합체의 평면 실장 형광 ...

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.

마우스 망막 색소 상피의 유전자 치료에 대한 바이러스 벡터의 윤부 접근 - 망막 주입

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 조회수.  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.

비디오: 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.

마우스 눈에 유전자 치료 벡터와 줄기 세포의 Subretinal 주입

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 ...

연구

의학

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.

나트륨 히알루 론산의 망막 주사 설치류에있는 망막 박리 모델

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.

서스펜션에 망막 색소 상피 세포를 제공하기 위해 설치류에서 망막 주사를 수행

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.

망막 하 공간에 액세스하기위한 대안의 검증 사출 방법<i&gt;를 통해</i&gt;를 Transcleral 후방 접근

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

신경과학

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.

쥐의 눈으로 인간 망막 안료 상피 세포의 트랜스 scleral Subretinal 이식에 대 한 세련 된 프로토콜의 개발

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.

초고 해상도 마우스 광학 일관성 단층 촬영 망막 유전자 치료 연구에서 안 구내 주사를 돕기 위해

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.

고양이과 거대 동물 모델에서 인간 배아 줄기 세포 유래 망막 조직의 망막하 이식

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.

작은 동물 모델에서 아데노 관련 바이러스 벡터의 결막하 투여

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.

난소 눈의 유리체 내 주사

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.

망막 변성 마우스 모델에서 망막하 이식편의 Transpupillary-Guided Trans-Scleral Transplantation

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