Hanlon Killian S, Cheng Ming, Ferrer Roberto Montoro, Ryu Jae Ryun, Lee Boram, De La Cruz Demitri, Patel Nikita, Espinoza Paula, Santoscoy Miguel C, Gong Yi, Ng Carrie, Nguyen Diane M, Nammour Josette, Clark Sean W, Heine Vivi M, Sun Woong, Kozarsky Karen, Maguire Casey A
Department of Neurology, Massachusetts General Hospital, Boston, MA 02115, USA; Molecular Neurogenetics Unit, Massachusetts General Hospital, Charlestown, MA 02129, USA; Harvard Medical School, Boston, MA 02116, USA; University College London, London, UK.
Department of Neurology, Massachusetts General Hospital, Boston, MA 02115, USA; Molecular Neurogenetics Unit, Massachusetts General Hospital, Charlestown, MA 02129, USA; Harvard Medical School, Boston, MA 02116, USA.
Mol Ther. 2024 Aug 7;32(8):2584-2603. doi: 10.1016/j.ymthe.2024.05.040. Epub 2024 Jun 5.
Systemic administration of adeno-associated virus (AAV) vectors for spinal cord gene therapy has challenges including toxicity at high doses and pre-existing immunity that reduces efficacy. Intrathecal (IT) delivery of AAV vectors into cerebral spinal fluid can avoid many issues, although distribution of the vector throughout the spinal cord is limited, and vector entry to the periphery sometimes initiates hepatotoxicity. Here we performed biopanning in non-human primates (NHPs) with an IT injected AAV9 peptide display library. We identified top candidates by sequencing inserts of AAV DNA isolated from whole tissue, nuclei, or nuclei from transgene-expressing cells. These barcoded candidates were pooled with AAV9 and compared for biodistribution and transgene expression in spinal cord and liver of IT injected NHPs. Most candidates displayed increased retention in spinal cord compared with AAV9. Greater spread from the lumbar to the thoracic and cervical regions was observed for several capsids. Furthermore, several capsids displayed decreased biodistribution to the liver compared with AAV9, providing a high on-target/low off-target biodistribution. Finally, we tested top candidates in human spinal cord organoids and found them to outperform AAV9 in efficiency of transgene expression in neurons and astrocytes. These capsids have potential to serve as leading-edge delivery vehicles for spinal cord-directed gene therapies.
将腺相关病毒（AAV）载体全身给药用于脊髓基因治疗存在诸多挑战，包括高剂量时的毒性以及会降低疗效的预先存在的免疫反应。将AAV载体鞘内（IT）注射到脑脊液中可避免许多问题，尽管载体在整个脊髓中的分布有限，并且载体进入外周有时会引发肝毒性。在这里，我们用IT注射的AAV9肽展示文库在非人灵长类动物（NHP）中进行了生物淘选。我们通过对从全组织、细胞核或转基因表达细胞的细胞核中分离的AAV DNA的插入片段进行测序来鉴定顶级候选物。这些带有条形码的候选物与AAV9混合，并比较其在IT注射的NHP的脊髓和肝脏中的生物分布和转基因表达。与AAV9相比，大多数候选物在脊髓中的保留增加。观察到几种衣壳从腰椎区域到胸椎和颈椎区域的扩散更大。此外，与AAV9相比，几种衣壳在肝脏中的生物分布减少，提供了高靶向/低脱靶生物分布。最后，我们在人脊髓类器官中测试了顶级候选物，发现它们在神经元和星形胶质细胞中转基因表达效率方面优于AAV9。这些衣壳有潜力作为脊髓定向基因治疗的前沿递送载体。
