A Biodistribution Study of OTV vs. ASO by Intravenous or Intrathecal Administration in Cynomolgus Monkeys

The objective of this study is to evaluate ASO biodistribution and pharmacodynamic response following intravenous delivery of conjugated OTV, which utilizes transferrin-receptor binding to deliver ASO, vs. intrathecal delivery of naked ASO in cynomolgus monkeys, the current gold standard for ASO delivery. Three groups were included for comparison: 1) Negative control cohort = Four biweekly intravenous doses of saline (n=3); 2) IT cohort = Single intrathecal dose of 4mg MALAT1 ASO (n=3); 3)  OTV multi dose cohort = Four biweekly intravenous doses of 30mpk OTV:MALAT1 (n=3). Tissues were collected two weeks after the final dose to allow sufficient time for target knockdown. Brain, spinal cord, and peripheral tissues were dissected and frozen for molecular analysis. Compared to an intrathecal injection of ASO, systemic administration of OTV resulted in significantly more homogenous biodistribution of ASO in the central nervous system as measured by a probe-based hybridization assay as well as staining with an anti-ASO antibody. One potential consequence of heterogeneous ASO distribution is heterogeneous target knockdown throughout the CNS. We observed more consistent target knockdown throughout the cortex, subcortical areas, and spinal cord of monkeys dosed peripherally with OTV. By contrast, monkeys dosed intrathecally with naked ASO showed much more knockdown in the spinal cord compared to the brian, consistent with ASO distribution. Single dose of 4mg MALAT1 ASO intrathecally (“ASO IT”), n=3 2)    Four biweekly doses of saline intravenously (“Saline” or “Vehicle”), n=3 3)    Four biweekly doses of 30mpk OTV:MALAT1 intravenously (“OTV IV”), n=3 Tissues were collected two weeks after the final dose to allow sufficient time for target knockdown. Brain tissue was dissected and frozen for pharmacodynamic analysis. Compared to an intrathecal injection of ASO, systemic administration of OTV resulted in significantly more widespread and homogenous biodistribution of ASO in the central nervous system as measured by staining with an anti-ASO antibody

本研究旨在评估利用转铁蛋白受体结合途径递送反义寡核苷酸（Antisense Oligonucleotide, ASO）的偶联OTV载体（Oligonucleotide Transport Vector, OTV）经静脉给药后的生物分布与药效学反应，并以当前ASO递送的金标准——裸ASO经鞘内给药的食蟹猴模型作为对照开展对比研究。
本研究共设置3组用于比较：1) 阴性对照队列：每两周一次静脉给予生理盐水，共给药4次，每组n=3；2) 鞘内给药队列：单次鞘内给予4mg MALAT1 ASO，每组n=3；3) OTV多剂量给药队列：每两周一次静脉给予30mpk的OTV:MALAT1偶联物，共给药4次，每组n=3。
于末次给药两周后采集组织，以获得充足的靶基因敲低时间。采集大脑、脊髓及外周组织并冷冻保存，用于后续分子分析。相较于ASO鞘内注射给药，通过基于探针的杂交实验以及抗ASO抗体染色检测结果显示，OTV的全身给药可使ASO在中枢神经系统（Central Nervous System, CNS）内呈现显著更均匀的生物分布。
ASO分布的异质性可能会导致中枢神经系统内靶基因敲低的异质性。本研究观察到，经外周途径给予OTV的食蟹猴，其大脑皮层、皮层下区域以及脊髓的靶基因敲低效果更为一致。与之形成对比的是，经鞘内给予裸ASO的食蟹猴，其脊髓中的靶基因敲低程度远高于大脑，这与ASO的组织分布特征相符。
本研究设置的3组具体为：1) 单次鞘内给予4mg MALAT1 ASO组（简称"ASO IT组"），n=3；2) 每两周一次静脉给予生理盐水组（简称"生理盐水组"或"赋形剂组"），n=3；3) 每两周一次静脉给予30mpk OTV:MALAT1组（简称"OTV IV组"），n=3。
于末次给药两周后采集组织，以获得充足的靶基因敲低时间。采集大脑组织并冷冻保存，用于药效学分析。相较于ASO鞘内注射给药，通过抗ASO抗体染色检测结果显示，OTV的全身给药可使ASO在中枢神经系统内呈现显著更广泛且均匀的生物分布。