Preclinical gene correction of hemophilia A

描述（由申请人提供）：该提案为血友病A基因治疗的临床试验奠定了基础。在这里，我们集中使用腺相关病毒（AAV）载体与新的衣壳血清型肝定向基因治疗的大型动物模型，即血友病A狗。这项工作建立在我们先前在两种新的AAV血清型AAV8和AAV9中使用FVIII基因递送“治愈”血友病A小鼠的成功基础上。我们在血友病A犬中的研究虽然较为成功，但也令人鼓舞地使用了两种新的AAV血清型。在本申请中，我们从重链和轻链载体中FVIII cDNA的递送开始，进一步评估这些AAV血清型。我们测试了AAV8和AAV9在两种不同剂量下的反应，并比较了载体施用的门静脉内途径和静脉内途径。然后，我们继续开发可以以高滴度产生的有效的、较小的单链载体，并使用血清型、剂量和施用途径的最佳组合测试该单链载体。我们还将在最佳条件下对幼犬进行治疗，以努力在严重发病率发生前开始治疗，并防止常规治疗中使用的rFVIII形成抑制剂。最后，我们将确定FVIII是否可以使用与原始病毒输注中使用的载体不同的血清型载体重新输注给犬。在所有这些研究中，将做出重大努力来测试新AAV载体的安全性，集中于中和抗体的研究、转氨酶的发展和病毒的生殖系传播。在这些研究结束时，我们希望发现在大型动物血友病A犬中持续产生长期治疗水平的FVIII（>正常值的10%）的治疗条件。这些结果应该使AAV载体的临床试验成为下一步的适当步骤。

DESCRIPTION (provided by applicant): This proposal sets the stage for clinical trials of gene therapy for hemophilia A. Here we concentrate on the use of adeno-associated viral (AAV) vectors with new capsid serotypes for liver directed gene therapy in a large animal model, namely, the hemophilia A dog. This work builds upon our previous success in "curing" the hemophilia A mouse using FVIII gene delivery in two new AAV serotypes, AAV8 and AAV9. Our studies in hemophilia A dogs, while more modestly successful, have also been encouraging with both new AAV serotypes. In this application, we evaluate further these AAV serotypes, beginning with delivery of FVIII cDNA in heavy chain and light chain vectors. We test responses at two different doses for both AAV8 and AAV9, and compare intraportal to intravenous routes of vector administration, We then go on to develop an effective, smaller single chain vector that can be produced in high titers, and test this single chain vector using the best combination of serotype, dose, and route of administration. We will also carry out therapy with our best conditions in young dogs, in an effort to initiate treatment before the onset of serious morbidity and also to prevent inhibitor formation to rFVIII used for conventional therapy. Lastly, we will determine whether FVIII can be readministered to dogs using a different serotype vector from the vector used in the original virus infusion. In all of these studies significant efforts will be made to test the safety of the new AAV vectors, concentrating on studies of neutralizing antibodies, development of transaminitis, and germline transmission of virus. At the end of these studies, we expect to have discovered treatment conditions that consistently produce long-term therapeutic levels of FVIII (>10% of normal) in a large animal, the hemophilia A dog. These results should make clinical trials with AAV vectors an appropriate next step.