Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
Division of Gastroenterology & Hepatology, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; Department of Medicine, University of Maryland Medical Center Midtown Campus, Baltimore, Maryland, USA.
Gastrointest Endosc. 2021 Dec;94(6):1119-1130.e4. doi: 10.1016/j.gie.2021.06.016. Epub 2021 Jun 29.
Gene therapy could provide curative therapies to many inherited monogenic liver diseases. Clinical trials have largely focused on adeno-associated viruses (AAVs) for liver gene delivery. These vectors, however, are limited by small packaging size, capsid immune responses, and inability to redose. As an alternative, nonviral, hydrodynamic injection through vascular routes can successfully deliver plasmid DNA (pDNA) into mouse liver but has achieved limited success in large animal models.
We explored hydrodynamic delivery of pDNA through the biliary system into the liver of pigs using ERCP and a power injector to supply hydrodynamic force. Human factor IX (hFIX), deficient in hemophilia B, was used as a model gene therapy.
Biliary hydrodynamic injection was well tolerated without significant changes in vital signs, liver enzymes, hematology, or histology. No off-target pDNA delivery to other organs was detected by polymerase chain reaction. Immunohistochemistry revealed that 50.19% of the liver stained positive for hFIX after hydrodynamic injection at 5.5 mg pDNA, with every hepatic lobule in all liver lobes demonstrating hFIX expression. hFIX-positive hepatocytes were concentrated around the central vein, radiating outward across all 3 metabolic zones. Biliary hydrodynamic injection in pigs resulted in significantly higher transfection efficiency than mouse vascular hydrodynamic injection at matched pDNA per liver weight dose (32.7%-51.9% vs 18.9%, P < .0001).
Biliary hydrodynamic injection using ERCP can achieve higher transfection efficiency into hepatocytes compared with AAVs at magnitudes of less cost in a clinically relevant human-sized large animal. This technology may serve as a platform for gene therapy of human liver diseases.
基因治疗可为许多遗传性单基因肝脏疾病提供治愈疗法。临床试验主要集中在腺相关病毒（AAV）用于肝脏基因传递。然而，这些载体受到小包装尺寸、衣壳免疫反应和无法再给药的限制。作为替代方案，通过血管途径的非病毒、流体动力学注射可以成功地将质粒 DNA（pDNA）递送到小鼠肝脏中，但在大型动物模型中仅取得了有限的成功。
我们探索了通过 ERCP 和动力注射器经胆道系统将 pDNA 递送到猪肝脏中的流体动力学递送，以提供流体动力。人凝血因子 IX（hFIX）是血友病 B 的缺陷基因，被用作基因治疗模型。
胆道流体动力学注射耐受性良好，生命体征、肝酶、血液学或组织学均无显著变化。聚合酶链反应未检测到其他器官的非靶向 pDNA 递送。免疫组织化学显示，在 5.5mg pDNA 的流体动力学注射后，50.19%的肝脏对 hFIX 呈阳性染色，所有肝叶的每个肝小叶均显示 hFIX 表达。hFIX 阳性肝细胞集中在中央静脉周围，向所有 3 个代谢区呈辐射状分布。在匹配的每肝重剂量 pDNA 时，猪胆道流体动力学注射比鼠血管流体动力学注射具有更高的转染效率（32.7%-51.9%比 18.9%，P<.0001）。
与 AAV 相比，使用 ERCP 的胆道流体动力学注射可以在成本较低的情况下实现更高的肝细胞转染效率，这在临床相关的大型人类动物中具有重要意义。该技术可以作为人类肝脏疾病基因治疗的平台。
