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。
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。
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。
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。?…
The authors have nothing to disclose.
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。
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 |