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.

在此PEGT的前四年中，我们进行了AAV介导的FVIII cDNA递送到血友病A小鼠和狗的基因。最近，我们发现使用新的AAV CAPSID血清型AAV8和AAV9，在两个单独的矢量或一个单个矢量中提供犬FVIII重型和轻链序列，从而可以完全校正小鼠的长期校正。该结果是通过术中或静脉内递送获得的。然而，犬内重链和轻链cDNA到AAV8载体中的狗A狗的犬内输送的结果令人失望。三只狗在18个月内部分校正了4-8％的活性，而另外两只只有1％的FVIII活性。由于FVIII基因疗法的最佳动物模型和最佳发育阶段尚不清楚，因此我们现在决定转向非人类的灵长类动物，恒河猴，并在胎儿和新生儿中进行基因递送。在特定目标1中，我们开发了猴子研究的工具，将人类FVIII cDNA克隆在两个单独的构建体中，并开发了人类特异性的FVIII抗体。在特定的目标2中，我们研究了两种不同剂量的向量，研究了三种不同AAV血清型AAV8，AAV9和最佳可用混合AAV载体的有效性。在此目标中，将对六个条件中的每一个评估两只胎儿猴子，所有胎儿在头三个月后期接受肝内载体的递送。在特定目标3中，我们将验证使用新生儿猴子和新生儿血友病A狗在特定目标2中发现的有效FVIII基因递送的最佳条件。因此，在这种续签中，我们将在早期发育中找到安全的长期FVIII表达的条件，以作为血友病的有用治疗选择的模型。