Kim Chi Heon, Oliver Colleen, Dar Hamid, Drissi Hicham, Presciutti Steven M
Department of Orthopaedics, Emory University School of Medicine, Atlanta, GA 30329, USA.
Atlanta Veteran Affairs Medical Center, Decatur, GA 30030, USA.
Genes Dis. 2020 Dec 30;9(4):1074-1085. doi: 10.1016/j.gendis.2020.12.009. eCollection 2022 Jul.
Intervertebral disc degeneration is the main contributor to low back pain, now the leading cause of disability worldwide. Gene transfer, either in a therapeutic attempt or in basic research to understand the mechanisms of disc degeneration, is a fascinating and promising tool to manipulate the complex physiology of the disc. Viral vectors based on the adeno-associated virus (AAV) have emerged as powerful transgene delivery vehicles yet a systematic investigation into their respective tropism, transduction efficiency, and relative toxicity have not yet been performed in the disc . Herein, we used bioluminescence imaging to systematically compare multiple AAV serotypes, injection volumes, titers, promoters, and luciferase reporters to determine which result in high transduction efficiency of murine nucleus pulposus (NP) cells . We find that AAV6 using a CAG promoter to drive transgene expression, delivered into the NP of murine caudal discs at a titer of 10 GC/mL, provides excellent transduction efficiency/kinetics and low toxicity . We also show, for the first time, that the transduction of NP cells can be significantly boosted by the use of small cell permeabilization peptides. Finally, to our knowledge, we are the first to demonstrate the use of optical tissue clearing and three-dimensional lightsheet microscopy in the disc, which was used to visualize fine details of tissue and cell architecture in whole intact discs following AAV6 delivery. Taken together, these data will contribute to the success of using AAV-mediated gene delivery for basic and translational studies of the IVD.
椎间盘退变是导致腰痛的主要原因，而腰痛目前是全球致残的首要原因。无论是在治疗尝试中还是在基础研究中，为了解椎间盘退变的机制而进行的基因转移，都是一种用于调控椎间盘复杂生理学的引人入胜且前景广阔的工具。基于腺相关病毒（AAV）的病毒载体已成为强大的转基因递送载体，但尚未在椎间盘中对它们各自的嗜性、转导效率和相对毒性进行系统研究。在此，我们使用生物发光成像系统地比较了多种AAV血清型、注射体积、滴度、启动子和荧光素酶报告基因，以确定哪些因素能导致小鼠髓核（NP）细胞的高转导效率。我们发现，使用CAG启动子驱动转基因表达的AAV6，以10¹² GC/mL的滴度递送至小鼠尾椎椎间盘的NP中，可提供优异的转导效率/动力学且毒性低。我们还首次表明，使用小分子细胞穿透肽可显著提高NP细胞的转导效率。最后，据我们所知，我们是首个证明在椎间盘中使用光学组织透明化和三维光片显微镜的研究，该技术用于在AAV6递送后可视化完整椎间盘中组织和细胞结构的精细细节。综上所述，这些数据将有助于AAV介导的基因递送在椎间盘基础研究和转化研究中取得成功。
