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，我们专注于在大型动物模型（即血友病a狗）中使用具有新衣壳血清型的腺相关病毒（AAV）载体进行肝定向基因治疗。这项工作建立在我们之前在两种新的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。我们对A型血友病犬的研究虽然比较成功，但对两种新的AAV血清型的研究也令人鼓舞。在本应用中，我们进一步评估这些AAV血清型，从在重链和轻链载体中递送FVIII cDNA开始。我们测试了AAV8和AAV9在两种不同剂量下的反应，并比较了入口内和静脉内的载体给药途径，然后我们继续开发一种有效的、较小的单链载体，可以产生高滴度，并使用血清型、剂量和给药途径的最佳组合来测试这种单链载体。我们还将在我们最好的条件下对幼犬进行治疗，努力在严重发病率发作之前开始治疗，并防止常规治疗中使用的rFVIII抑制剂形成。最后，我们将确定是否可以使用不同于原始病毒输注载体的血清型载体重新给犬接种FVIII。在所有这些研究中，将作出重大努力来测试新的AAV载体的安全性，集中研究中和抗体、转氨炎的发展和病毒的种系传播。在这些研究结束时，我们期望在大型动物血友病a犬中发现持续产生长期治疗水平的FVIII的治疗条件（bbb10正常值的10%）。这些结果将为下一步使用AAV载体进行临床试验提供依据。

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.