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
Summary
在这里, 我们展示了一种新的方法, 使用高分辨率光谱域光学相干断层扫描 (hr-sd-oct), 以协助将基因治疗剂传递到视网膜下空间, 评估其区域覆盖, 并表征光感受器的活力。
Abstract
hr-sd-oct 用于监测活小鼠模型中光感受器变性的进展, 评估治疗药物在视网膜下空间的传递, 并评估体内的毒性和有效性。hr-sd-oct 使用近红外光 (800-880 nm), 并具有专为鼠标眼睛独特光学器件设计的光学器件, 其轴向分辨率低于2微米。对转基因小鼠外视网膜 (光感受器) 变性和对照进行了成像, 以评估疾病进展情况。采用带状玻璃微针, 通过反层和反式脉络膜法,对腺病毒 (aav) 或纳米颗粒 (np) 进行视网膜下注射。在校准压力注射之前, 需要将针头仔细定位到视网膜下空间, 然后将液体输送到视网膜下空间。在视网膜成像系统 (ris) 上进行了实时视网膜下手术。hr-sd-oct 表现出由于毒性突变体人视紫红质 (p347s) (rhop347s) 转基因表达而逐渐变性视网膜变性。hr-sd-oct 允许对所有视网膜层进行严格的定量。外核层 (onl) 厚度和光感受器外段长度 (osl) 测量与光感受器的活力、退化或抢救相关。ris 分娩系统可实时显示新生儿 (~ p10-14) 或成年小鼠的视网膜下注射, hr-sd-oct 立即确定分娩的成功程度和地图面积。hr-sd-oct 是一个强大的工具, 可以评估小鼠视网膜下手术的成功, 以及测量光感受器在体内的活力。hr-sd-oct 还可用于识别统一的动物队列, 以评估视网膜退化的程度、毒性和治疗性抢救在临床前基因治疗研究中的应用。
Introduction
研究人员正在开发各种视网膜和视网膜退行性疾病的基因疗法, 希望将新的疗法转化为人类疾病的治疗方法1,2,3,4,5,6,7.,8,9,10,11. 时域或光谱域光学相干断层扫描 (sd-oct) 已被用来研究疾病 12、13、14的特定小鼠模型中视网膜外变性的各个方面.然而, hr-sd-oct 并没有被广泛用于优化小鼠模型的评价, 以确定视网膜变性的速度和空间一致性, 或者在基于基因的疗法的临床前评估中, 例如:评估救援、毒性或病媒传递的空间范围8、15、16。一旦小鼠模型被充分描述, hr-sd-oct 数据可以作为一个信息丰富和可靠的资源, 以衡量治疗方法的影响, 发挥救援或毒性的小鼠模型视网膜变性 17。许多群体使用视网膜下注射作为载体传递的方法, 因为它在转染光感受器和视网膜色素上皮 (rpe) 细胞的效率。然而, 这仍然是一个很难掌握的方法, 因为它通常是通过从角膜表面的自由手手术, 往往充满白内障, 出血, 和意外视网膜脱离发生仅仅是通过操纵后部玻璃体。许多群体仍然试图盲目地注射视网膜下注射, 并使用人工注射相对较大直径的不锈钢针头 (34g)8,17,18,19 提供病毒 20,21, 22,和一些使用光学相干断层扫描 (oct) 成像, 以确认适当的载体传递到视网膜8,17,20,22. 最近使用微机械手22驱动的微型针头描述了该方法的一些改进.
我们提出了一个综合的方法, 帮助在针的定位, 注射是由一个定制的定向立体眼镜仪设计在实验室专门为可视化内的小鼠17,23. 在使用拉玻璃微针头与立体定向微机械手相结合的情况下, 可以更好地控制针头的放置, 无需手术切除 (即通过结膜和结缔组织)。注射。使用压力调节的微注射器有助于提供一致的注射量, 并且与手持式注射器进行的手动注射相比, 注射的稳定性、精度和速度要慢得多, 从而降低了出现泡沫注射到眼睛。较小的针头有助于防止拔针后泄漏, 因为路径是自密封的。为了评估注射的程度, 许多研究小组依靠在实验结束时发现和评估视网膜中增强的绿色荧光蛋白 (egfp) 表达的区域范围 (由载体提供的表达结构)点 (安乐死) 确认注射成功11,19,20,24。这种方法 (不使用 oct) 来验证手术的成功, 浪费了大量的资源在外科手术时间和动物, 因为所有动物有 (未知) 手术失败需要保持, 其次是重复措施, 直到安乐死和眼睛收获 (当 egfp 被测量)。使用 hr-sd-oct 可以改善注射在视网膜中位置的确认, 以证明注射位于视网膜的正确层 (即视网膜下空间) 之间。hr-sd-oct 也可用于立即描述不成功的尝试 (手术失败), 以确定相关变量在实际手术时间, 以改进的方法。我们发现, HR-SD-OCT 通过允许快速定量评估视网膜外变性, 从而在临床前基因治疗研究中提供了许多优势, 从而能够识别不符合实验标准的研究动物 (例如,不正确的视网膜下注射), 并直接随访成像眼睛的区域, 其中矢量的交付 (在临床前的效果是最可能的), 以及控制区域, 其中矢量没有交付。自发展以来, sd-oct 的使用一直被眼科研究人员接受和使用, 现在被认为是小鼠或啮齿类动物模型 13,25视网膜科学研究中视网膜成像的标准。hr-sd-oct 及其软件能力以独特的综合方式被利用, 以促进小鼠模型在这一过程中的每一步成功进行定量基因治疗的目标, 包括动物模型的选择、选择中的退化特征疾病模型, 治疗传递, 病媒传递的映射, 和毒性/疗效评估。hr-sd-oct 的使用可以在整个过程的各个层面更有效地发现药物。在这里, 我们描述了这些方法, 用于我们的 rna 药物发现计划。
Protocol
Representative Results
Discussion
hr-sd-oct 为人类疾病潜在动物模型的定性提供了一种简单的方法, 以确定它们在测试潜在疗法方面的有用性。快速可靠地描述人类疾病潜在动物模型的能力对于治疗药物发现的过程至关重要 (例如, 替代基因治疗、核酶或 shnna 敲除基因治疗、联合基因治疗)。hr-sd-oct 为评估视网膜健康提供了一种简单、快速和非侵入性的方法, 可用于描述和监测几乎所有小鼠模型中视网膜退化的进展。oct 图像可…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这些材料的部分依据是退伍军人事务部、退伍军人卫生管理局、研究与发展办公室 (生物医学实验室研究与开发) (退伍军人健康研究所) (退伍军人健康研究所赠款 1i01 bx000669) 所支持的工作。jms 部分由 va wny 聘为眼科工作人员医生-科学家;mcb 部分受雇于 va wny。这项研究是在纽约西部医疗系统 (纽约州布法罗) 进行的, 并得到了部分支持。内容不代表退伍军人事务部或美国政府的意见。还在很大程度上支持, NIH/NEI 授予 ey013433 (p:jms), nih变 nei r24 授予 ey016662 (ub 视觉基础设施中心, pi: m 屠宰, 目录-biophotonics 模块: jms), 向奥曼诺林大学系提供不受限制的赠款布法罗来自预防失明研究 (纽约州纽约), 以及 oishei 基金会 (纽约州布法罗) 提供的一笔赠款。我们承认 hc1 转基因rhop347s线的礼物和来自 janis lem 博士 (波士顿塔夫茨新英格兰医疗中心) 的 exon 1老鼠 rho淘汰赛的礼物, 以及 nrt-e 转基因模型在杂合状态下的天赋。鼠标外显子 2 rho 淘汰赛背景从g. 简·法拉尔博士和彼得亨弗里斯博士 (三一学院, 都柏林, 爱尔兰共和军)。
Materials
| C57BL6 (J) | Jackson Laboratories | 664 | |
| N129R- | N/A | N/A | |
| 2HRho 1T/1T | N/A | N/A | |
| Envisu R-2200 Ocular Coherence Tomography Instrument (OCT) | Bioptigen | 90-R2200-U1-120. | |
| Retinal Imaging System (RIS) | In-house | N/A | |
| Stemi 2000C Microscope | Zeiss | 000000-1106-133 | |
| P-97 Flaming/Brown micropipette puller | Sutter Instrument Co | p-97 | |
| MMN-33 micro manipulator | Narishige USA | MMN-33 | |
| PLI-100 micro injector | Harvard Apparatus | 64-1736 | |
| Micropipette Holder (Rotating) | In-house | N/A | |
| Micropipette Storage Receptacle | World Precision Instruments Inc. | E210 | |
| Borosilicate glass capillary tubes 1.5-1.8 X 100mm, | Harvard Apparatus | 30-0053 | |
| 2,2,2-Tribromoethanol | SIGMA Aldrich | T48402-25G | |
| Tert-amyl Alcohol | SIGMA Aldrich | 240486-100ML | |
| Atropine Sulfate Ophthalmic Solution, USP 1% | Akorn Inc. | NDC 17478-215-05 | |
| Goniovisc | BioVision Limited | NDC 17238-610-15 | |
| Cyclopentolate Hydrochloride Ophthalmic Solution, USP 2% | Akorn Inc. | NDC 17478-097-10 | |
| Gentamicin Sulfate Ointment USP, 0.1% | Perigo | NDC 45802-046-35 | |
| Systane Ultra | Alcon Laboratories, Inc. | 9006619-1013 | |
| Tetracaine Hydrochloride | Bausch and Lomb | NDC 24208-920-64 | |
| Ophthalmic Solution, USP 0.5% | Bausch and Lomb | NDC 24208-920-64 | |
| DPBS | Gibco Life Technologies | 14190-136 | |
| Virus Preparations | ViGene /UNC | N/A | |
| Gold nanorods | NANOPARTz | D12M-850-1.75 | |
| Fluorescein Sulfate AK-FLUOR 25% | Akorn Inc. | NDC 17478-250-20 | |
| Coverslips | Fisher Scientific | 12-548-A | |
| Forceps | Milton | 18-825 | |
| Needles 30 guage | Beckton Dickenson | W11604 | |
| Syringes | Beckton Dickenson | 309659 | |
| Bioptigen software Package | Bioptigen | N/A | |
| Proparacaine Hydrochloride Ophthalmic Solution, USP 0.5% | Akorn Inc. | NDC 17478-263-12 | |
| Windows Excel | Microsoft | N/A | |
| Adobe Illustrator | Adobe | ||
| Scale | Mettler | ||
| Scissors | World Precision Instruments | ||
| Ear punch | Nat’l band | ||
| CL 100 Light source | Welch Allyn | CL100 | |
| Nitrogen Gas | Jackson Welding Supply | N/A | |
| Heated Water bath | Neslab | RTE-140 | |
| Heating plate | In House | N/A | |
| Heating mat | Cincinnatti Sub Zero | 273 | |
| Clay mouse holder | Plast.i.clay American Art Clay Co. | N/A | |
| Betadine | MedLine | NDC53329-938-06 | |
| Cotton Tip Applicators | American Health Service | Ctag | |
| EtOH 70% | Fisher Scientific | BP2818-100 | |
| Gloves Nitrile | VWR | 89038-272 | |
| Diagnosys ERG Color Dome instrument | Diagnosys Inc. | D125 | |
| Contact lenses | In-house | N/A | |
| Diagnosys Software | Diagnosys Inc. | N/A | |
| Origin 6.1 software | OriginLab Corp. | N/A | |
| Reference electrodes | Ocuscience | F-Thread Electrode (DTL) 24” |
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