Preclinical Development of an AAV8-hUGT1A1 Vector for the Treatment of Crigler-Najjar Syndrome
Preclinical Development of an AAV8-hUGT1A1 Vector for the Treatment of Crigler-Najjar Syndrome
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DOI：
10.1016/j.omtm.2018.12.011
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发表时间：
2019-03-15
影响因子：
4.7
通讯作者：
Mingozzi, Federico
中科院分区：
文献类型：
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作者：
Collaud, Fanny;Bortolussi, Giulia;Mingozzi, Federico
腺相关病毒（Adeno-associated viruses，AAV）是肝脏基因治疗中最有效的载体之一。在第一次血友病临床试验中获得的结果证明了这种方法在人类中的长期疗效，显示了用自身互补（sc）和单链（ss）AAV载体有效靶向肝细胞。然而，为了支持基于AAV的基因疗法的临床开发，需要高效且可扩展的生产工艺。为了将AAV介导的肝脏基因转移方法转化为临床治疗Crigler-Najjar（CN）综合征，我们开发了一种（ss）AAV 8载体，其携带在肝脏特异性启动子控制下的人UDP-葡萄糖醛酸基转移酶家族1成员A1（hUGT 1A 1）转基因。我们将我们的构建体与表达hUGT 1A 1的类似（sc）AAV 8载体进行了比较，显示出相当的体外和体内效力。相反，（ss）AAV 8-hUGT 1A 1载体与它们的（sc）对应物相比显示出上级的产率和产物均一性。然后，我们将我们的努力集中在基于悬浮培养的HEK 293细胞的三重转染的临床产品（ss）AAV 8-hUGT 1A 1的制造过程的规模扩大上。该载体的大规模生产具有与在贴壁细胞中生产的小规模载体相同的特征。还使用大规模的载体制剂对该疾病的动物模型进行了临床前研究，并进行了良好实验室规范毒理学-生物分布研究。这些研究证明了在该疾病的相关动物模型中用（ss）AAV 8-hUGT 1A 1进行基因转移的长期安全性和有效性，从而支持这种基因治疗方法用于治疗CN综合征的临床转化。
Adeno-associated viruses (AAVs) are among the most efficient vectors for liver gene therapy. Results obtained in the first hemophilia clinical trials demonstrated the long-term efficacy of this approach in humans, showing efficient targeting of hepatocytes with both self-complementary (sc) and single-stranded (ss) AAV vectors. However, to support clinical development of AAV-based gene therapies, efficient and scalable production processes are needed. In an effort to translate to the clinic an approach of AAV-mediated liver gene transfer to treat Crigler-Najjar (CN) syndrome, we developed an (ss) AAV8 vector carrying the human UDP-glucuronosyltransferase family 1-member A1 (hUGT1A1) transgene under the control of a liver-specific promoter. We compared our construct with similar (sc) AAV8 vectors expressing hUGT1A1, showing comparable potency in vitro and in vivo. Conversely, (ss) AAV8-hUGT1A1 vectors showed superior yields and product homogeneity compared with their (sc) counterpart. We then focused our efforts in the scale-up of a manufacturing process of the clinical product (ss) AAV8-hUGT1A1 based on the triple transfection of HEK293 cells grown in suspension. Large-scale production of this vector had characteristics identical to those of small-scale vectors produced in adherent cells. Preclinical studies in animal models of the disease and a good laboratory practice (GLP) toxicology-biodistribution study were also conducted using large-scale preparations of vectors. These studies demonstrated long-term safety and efficacy of gene transfer with (ss) AAV8-hUGT1A1 in relevant animal models of the disease, thus supporting the clinical translation of this gene therapy approach for the treatment of CN syndrome.