绵羊眼玻璃体内注射
Summary
在羊眼中进行玻璃体内注射，目的是将病毒介导的基因治疗传递到视网膜。
Abstract
有几种方法可以将治疗剂输送到视网膜，包括玻璃体内 （IVT）、视网膜下、脉络膜上、眼周或局部给药。IVT药物输送涉及注射到眼睛的玻璃体中，玻璃体是一种充满眼睛后房并保持眼球形状的凝胶状物质。虽然IVT途径的特异性目标性不如视网膜下递送，但它的侵入性要小得多，并且在临床环境中广泛用于一系列眼部疾病。
我们之前证明了玻璃体内递送腺相关病毒（AAV）介导的基因治疗产物（AAV9.CLN5）在绵羊中具有天然存在的CLN5形式的神经元蜡样脂褐质沉着症（NCL）。受影响的绵羊一只眼睛接受IVT基因治疗，另一只未经治疗的眼睛作为内部对照。治疗后15个月内，接受治疗的眼睛保持视网膜结构和功能，而未经治疗的眼睛在尸检时表现出进行性下降和严重萎缩。根据绵羊研究，CLN5基因治疗产品于2021年9月被美国食品药品监督管理局批准为候选研究新药（IND）。本文详细介绍了IVT将治疗性病毒载体递送到绵羊眼的手术方案。
Introduction
有几种方法可用于将治疗剂输送到视网膜，包括玻璃体内 （IVT）、视网膜下、脉络膜上、眼周或局部给药。每种给药途径都涉及克服诸如血液 – 视网膜屏障或内部和外部限制膜之类的屏障，并且根据所输送的药物和特定的视网膜靶标1，2具有不同的疗效率。
IVT药物输送涉及将注射到眼睛的玻璃体中，玻璃体是一种占据眼睛后房的凝胶状物质。玻璃体的主要功能是维持眼球的形状并保持眼组织（如晶状体和视网膜）就位。玻璃体液主要由水组成，少量胶原蛋白、透明质酸和其他非胶原蛋白3。IVT注射是一种简单而常见的程序，常规用于治疗各种眼部疾病，包括年龄相关性黄斑变性，糖尿病黄斑水肿，糖尿病视网膜病变，视网膜静脉阻塞和几种遗传性视网膜营养不良4，5。
神经元蜡样脂褐素糖（NCL;巴顿病）是一组致命的溶酶体贮积病，会导致大脑和视网膜严重退化。目前有13种已知的NCL变异，由不同基因（CLN1-8，CLN10-14）的突变引起，主要影响儿童，但发病年龄和疾病严重程度为6。NCL具有共同的进行性症状，包括认知和运动衰退，癫痫发作和视力丧失。NCL无法治愈;然而，脑定向酶替代疗法目前正在针对CLN2疾病7，8进行临床试验，AAV介导的基因疗法在临床前研究中显示出巨大的前景，CLN5基因治疗的临床试验预计将于2022年开始9，10。
许多其他物种发展出天然存在的NCL形式，包括猫，狗，羊和牛。NCL的两种绵羊模型目前正在新西兰积极研究中：Borderdale绵羊的CLN5疾病模型和南汉普郡绵羊的CLN6疾病模型。受影响的绵羊表现出人类疾病的许多临床和病理特征，包括视网膜萎缩和视力丧失10，11。尽管在患有CLN5疾病的绵羊中进行脑定向CLN5基因治疗可以预防或阻止脑萎缩和临床衰退，但接受治疗的绵羊仍然会失去视力9。这突出了治疗视网膜以保护视力和保持更好生活质量的必要性，从而建立了绵羊眼部基因治疗方案。
羊眼代表了人眼的良好模型，因为它在眼球尺寸、玻璃体体积和视网膜结构10、12、13 方面相似。本文详细介绍了IVT将小体积（≤100μL）治疗性病毒载体输送到羊眼的手术方案。
Protocol
所有实验方案均由林肯大学动物伦理委员会批准，并符合美国国立卫生研究院关于在研究中照顾和使用动物的指南和新西兰动物福利法案（1999）。边境代尔绵羊在14 岁出生时被诊断出来，并在林肯大学的研究农场饲养。三只3个月大的纯合子（CLN5-/-）母羊接受了左眼单次IVT注射，未经治疗的右眼作为内部对照。将视网膜电图和病理学数据与历史健康和受影响的对照数据?…
Representative Results
该研究小组先前已证明 IVT 递送 CLN5 基因治疗载体在减轻 CLN5 NCL 绵羊视网膜功能障碍和变性的功效15.受影响的绵羊接受单次 100 μL IVT 注射包装在 AAV 血清型 9 （AAV9） 载体 （AAV9.CLN5）进入一只眼睛，对侧眼睛作为未经治疗的内部对照。从注射年龄（3个月）到终末期疾病（18个月）每月评估视力。对治疗和未治疗的眼睛以及年龄匹配的健康和受CLN5影响的对照组进行了视网膜组织学…
Discussion
玻璃体内注射是人类眼科中最常见的外科手术之一，已被证明可有效将AAV介导的基因疗法输送到绵羊的视网膜。我们之前已经证明了AAV9的功效。CLN5基因治疗在玻璃体内使用CLN5 NCL15减轻绵羊视网膜功能障碍和变性。希望将这种给药途径转化为人类NCL患者也将证明是有益的。
小容量IVT注射到羊眼的方案相对简单和非侵入性，易于重现，并且易于非专家学习。?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
作者要感谢Steve Heap博士（BVSc，CertVOphthal）在建立该协议和执行Murray等人描述的注射方面的帮助15。作者还感谢来自CureKids New Zealand，Canterbury医学研究基金会，Neurogene Inc和Batten Disease Support and Research Association的资助。
Materials
| 1 mL low dead-space safety syringe with permanently attached 0.5 inch needle | Fisher Scientific, Auckland, New Zealand | 05-561-28 | Covidien Monoject Tuberculin Safety syringe or similar |
| 1.5 mL microcentrifuge tube | Sigma Aldrich | HS4323 | Autoclave tubes to sterilise prior to use |
| Anesthesia machine with gas bench and monitor | Hyvet Anesthesia, Christchurch, New Zealand | ||
| Antibiotic eye drops | Teva Pharma Ltd, Auckland, New Zealand | Commercial name: Chlorafast (0.5% chloramphenicol) | |
| BrightMount plus anti-fade mounting medium | Abcam, Cambridge, United Kingdom | ab103748 | |
| DAPI (4′ ,6-diamidino-2-phenylindole dihydrochloride) | Sigma Aldrich, St. Louis, Missouri, United States | 10236276001 | |
| Diazepam sedative | Ilium, Troy Laboratories Pty Ltd, Tauranga, New Zealand | 5 mg/mL | |
| Endotracheal tubes | Flexicare Medical Ltd, Mountain Ash, United Kingdom | Standard, cuffed. Sizes 7, 7.5, or 8 depending on sheep size | |
| Eye speculum | Capes Medical Ltd, Tauranga, New Zealand | KP151/14 | Nopa Barraquer-Colibri (10 mm) |
| Fenestrated surgical drape | Amtech Medical Ltd, Whanganui, New Zealand | DI583 | Or similar |
| Filter Tips | Interlab, Auckland, New Zealand | 10, 200, and 1,000 µL | |
| Formaldehyde solution (37%) | Fisher Scientific, Auckland, New Zealand | AJA809-2.5PL | Make up to 10% in distilled water with 0.9% NaCl |
| Goat anti-rabbit Alexa Fluor 594 | Invitrogen Carlsbad, CA, USA | A-11012 | Use at a dilution of 1:500 |
| Isoflurane anesthetic | Attane, Bayer Animal Health, Auckland, New Zealand | ||
| Ketamine HCl anesthetic/analgesic | PhoenixPharm Distributors Ltd, Auckland, New Zealand | 100 mg/mL | |
| Laryngoscope (veterinary) | KaWe Medical, Denmark | Miller C blade, size 2 | |
| Needles | Capes Medical Ltd, Tauranga, New Zealand | 302025 | BD Hypodermic Needles, or similar |
| Non-steroidal anti-inflammatory | Boehringer Ingelheim (NZ) Ltd, Auckland, New Zealand | 49402/008 | Commercial name: Metacam 20 (20 mg/mL meloxicam) |
| Non-toothed forceps | Capes Medical Ltd, Tauranga, New Zealand | AB864/16 | Or similar |
| Non-toothed hemostat | Capes Medical Ltd, Tauranga, New Zealand | AA150/12 | Or similar |
| Normal goat serum | Thermo Fisher Scientific, Christchurch, New Zealand | 16210072 | |
| Oxygen (medical) | BOC Gas, Christchurch, New Zealand | D2 cylinder, gas code 180 | |
| Phosphate buffered saline | Thermo Fisher Scientific, Christchurch, New Zealand | 10010023 | Sterile, filtered |
| Povidone-Iodine solution | Capes Medical Ltd, Tauranga, New Zealand | 005835 | Commercial name: Betadine (10% povidone-iodine) |
| Rabbit anti-cow glial fibrillary acidic protein (GFAP) | Dako, Glostrup, Denmark | Z0334 | Use at a dilution of 1:2,500 |
| Self-complementary adeno-associated virus serotype 9, containing the chicken beta action (CBh) promoter and codon-optimized ovine CLN5 | University of North Carolina Vector Core, NC, USA. | scAAV9/CBh-oCLN5opt | |
| Sodium Chloride 0.9% IV Solution | Capes Medical Ltd, Tauranga, New Zealand | AHB1322 | Commercial name: Saline solution |
| Subcutaneous antibiotics | Intervet Schering Plough Animal Health Ltd, Wellington, New Zealand | Commercial name: Duplocillin LA (150,000 IU/mL procaine penicillin and 115,000 IU/mL benzathine penicillin) | |
| Surgical sharp blunt curved scissors | Capes Medical Ltd, Tauranga, New Zealand | SSSHBLC130 | |
| Terumo Syringe Luer Lock | Amtech Medical Ltd, Whanganui, New Zealand | SH159/SH160 | Sterile syringes; 10 mL for drawing up induction drugs, 20 mL for drawing up saline |
| Virkon Disinfectant Powder | EBOS Group Ltd, Christchurch, NZ | 28461115 |
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Citazione di questo articolo
Murray, S. J., Mitchell, N. L. Intravitreal Injections in the Ovine Eye. J. Vis. Exp. (185), e63823, doi:10.3791/63823 (2022).