Intravascular Delivery of AAV to Skeletal Muscle

在以前的工作中，我们发现肌肉注射表达F.IX的重组腺相关病毒(AAV)2型载体可以导致F.IX在小鼠和血友病犬体内的长期表达。第一阶段临床研究表明，肌肉注射AAV-F.IX在人体内是安全的，并且通过所有受试者注射肌肉的活组织检查判断，导致了基因转移和表达。尽管直接肌注很容易获得骨骼肌，但实现AAV治疗目标剂量 人类已经被证明是不切实际的，因为需要大量的注射(约300个部位)。因此，人们对开发无需数百次肌肉注射即可转导大量肌肉纤维的技术非常感兴趣。骨骼肌包含丰富的毛细血管网络，可以通过化学和/或机械修饰来确保含有载体的液体的血管渗漏。这项工作的总体目标是建立新型区域血管内载体载体到肢体骨骼肌广泛区域的有效性和安全性。在目标1中，我们将确定两种不同的区域血管内给药方法的有效性和安全性。第一种方法是隔离肢体灌流(ILP)，其中载体通过股动脉输送。第二种方法是顺行肢体灌注(ALP)，通过血管远端浅静脉注射载体。 在静水压力升高的情况下的肢体。这一目标受到了我们关于这些技术的初步数据的鼓舞，这些数据记录了正常狗后肢肌肉组织的广泛转导，以及在严重血友病B的犬模型中血友病B表型的改善。在目标2中，我们将确定不同血清型的AAV载体是否会导致F.IX表达水平更高。这些结果将在以下方面具有临床意义：(A)非AAV-2载体的剂量-反应优势，以及(B)治疗AAV-2高滴度中和抗体患者(约占总人口的20%)。在目标3中，我们将测试F.IX变体的使用是否可以在F.IX的量方面提高疗效 是纠正血友病B表型所必需的。对于一种疾病类别，其中相当大比例(80%)的严重影响的成年人有医源性潜在的肝病，肌肉导向策略是非常有吸引力的。这项转译研究的结果将为血友病B患者的这种传递方法的临床研究以及肌肉营养不良等其他疾病的基因转移奠定实验基础。

In previous work, we showed that intramuscular (IM) injection of a recombinant adeno-associated viral (AAV) serotype 2 vector expressing F.IX resulted in long-term expression of F.IX in mice and hemophilic dogs. Phase I clinical studies demonstrated that IM injection of AAV-F.IX in humans was safe and resulted in gene transfer and expression as judged by biopsy of injected muscle in all patients tested. Although access to skeletal muscle is easily performed by direct IM injections, achievement of AAV therapeutic target doses in humans has proved impractical because of the large number (> 300 sites) of injections required. Thus there is a great interest in exploiting techniques that allow transduction of large numbers of muscle fibers without requiring hundreds of intramuscular injections. Skeletal muscle contains a rich network of capillaries that can be chemically and/or mechanically modified to ensure vascular leakage of fluid containing vectors. The overall goal of this work is to establish the efficacy and safety of novel regional intravascular delivery methods of vectors to extensive areas of the skeletal muscle of a limb. In aim 1 we will determine the efficacy and safety of two different regional intravascular delivery methods. The first method is isolated limb perfusion (ILP) in which the vector is delivered through the femoral artery. The second approach is anterograde limb perfusion (ALP) in which the vector is injected through a superficial vein in the distal part of the limb under elevated hydrostatic pressure. This goal is encouraged by our preliminary data on these techniques documenting widespread transduction of muscle tissue of the hind limb in normal dogs and by the improvement of the hemophilia B phenotype in a canine model of severe hemophilia B. In aim 2 we will determine whether AAV vectors of alternate serotypes result in higher levels of F.IX expression. These results will have clinical implications in terms of (a) dose-response advantage of non-AAV-2 vectors, and (b) treatment of patients with high titer neutralizing antibodies to AAV-2 (about 20% of general population). In aim 3 we will test whether the use of F.IX variants could improve the efficacy in terms of the amount of F.IX required for the correction of the hemophilia B phenotype. For a disease category in which a substantial percentage (>80%) of severely affected adults have iatrogenic underlying liver disease, a muscle-directed strategy is very attractive. The results of this translational study will establish an experimental basis for clinical studies of this delivery method in humans with hemophilia B and also for gene transfer for other diseases such as muscular dystrophy.