Model Gene Therapy of Hemophilia A via Liver Directed

通过肝脏定向的 A 型血友病基因治疗模型

• 批准号: 7154967
• 负责人: Haig Kazazian
• 金额: $ 19.38万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-29 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7154967
• 关键词: Macaca mulatta; adeno associated virus group; antibody; biotechnology; clotting factor; complementary DNA; cooperative study; disease /disorder model; dogs; embryo /fetus; gene delivery system; gene therapy; genetic strain; hemophilia As; hemophilia B; liver; model design /development; molecular cloning; recombinant proteins; transfection /expression vector

In the previous four years of this PEGT, we carried out AAV-mediated gene delivery of FVIII cDNA to hemophilia A mice and dogs. Recently, we found that use of the new AAV capsid serotypes, AAV8 and AAV9, to deliver canine FVIII heavy and light chain sequences in either two separate vectors or a single vector led to complete long-term correction of hemophilia A mice. This result was obtained with either intraportal or intravenous delivery. However, results from intraportal delivery of canine heavy and light chain cDNAs to hemophilia A dogs in an AAV8 vector have been disappointing. One dog in three has had partial correction of 4-8% activity for 18 months, while the other two had only 1% FVIII activity. Since the best animal model and best developmental stage for FVIII gene therapy are unknown, we now have decided to turn to a non-human primate, the rhesus macaque, and to carry out gene delivery in the fetus and neonate. In Specific Aim 1, we develop the tools for monkey studies, cloning human FVIII cDNA in two separate constructs and developing a human-specific FVIII antibody. In Specific Aim 2, we study the effectiveness of three different AAV serotypes, AAV8, AAV9, and the best available hybrid AAV vector, at two different doses of vector. In this Aim, two fetal monkeys will be evaluated for each of the six conditions with all fetuses undergoing intrahepatic delivery of vector in late first trimester. In Specific Aim 3, we will validate the optimal conditions for effective FVIII gene delivery discovered in Specific Aim 2 using neonatal monkeys and neonatal hemophilia A dogs. Thus, in this renewal we will find conditions for safe, long term FVIII expression in early development as a model for a useful treatment option for hemophilia A.

在该 PEGT 的前四年中，我们对 A 型血友病小鼠和狗进行了 AAV 介导的 FVIII cDNA 基因递送。最近，我们发现使用新的 AAV 衣壳血清型 AAV8 和 AAV9 在两个单独的载体或单个载体中传递犬 FVIII 重链和轻链序列，可以完全长期纠正血友病 A 小鼠。该结果是通过门静脉内或静脉内递送获得的。然而，通过 AAV8 载体向 A 型血友病狗门静脉内递送犬重链和轻链 cDNA 的结果令人失望。三分之一的狗在 18 个月内部分校正了 4-8% 的活性，而另外两只狗的 FVIII 活性仅为 1%。由于FVIII基因治疗的最佳动物模型和最佳发育阶段尚不清楚，我们现在决定转向非人类灵长类动物——恒河猴，并在胎儿和新生儿中进行基因传递。在具体目标 1 中，我们开发了用于猴子研究的工具，将人 FVIII cDNA 克隆到两个单独的构建体中，并开发了人特异性 FVIII 抗体。在具体目标 2 中，我们研究了三种不同 AAV 血清型（AAV8、AAV9）和最佳可用混合 AAV 载体在两种不同剂量载体下的有效性。在这个目标中，将评估两只胎儿猴的六种情况中的每一种，所有胎儿在妊娠早期末接受载体的肝内递送。在特定目标 3 中，我们将使用新生猴和新生 A 型血友病犬验证特定目标 2 中发现的有效 FVIII 基因递送的最佳条件。因此，在本次更新中，我们将找到早期发育中安全、长期 FVIII 表达的条件，作为 A 型血友病有用治疗选择的模型。