摘要
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 in vivo.Herein,we used in vivo 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 in vivo.We find that AAV6 using a CAG promoter to drive transgene expression,delivered into the NP of murine caudal discs at a titer of 1011 GC/mL,provides excellent transduction efficiency/kinetics and low toxicity in vivo.We also show,for the first time,that the transduction of NP cells can be significantly boosted in vivo 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.
基金
This work was supported by a Veteran Affairs Career Development Award(IK2-BX003845).
