Gene correction for Hemophilia A

这项建议为血友病A的基因治疗的临床试验奠定了基础，同时启动了对新载体系统的研究。我们最初将在我们的小鼠模型中确定最有效和最安全的因子VIII(FVIII)缺乏症的基因纠正策略。我们之前已经证明，在转基因小鼠和皮肤外植体中，功能性VIII可以从基底上表皮传递到循环中。我们将通过使用1)抗灭活的FVII和2)物种特有的FVIII来改进Fvii的交付。然后，我们使用最优的FVIII结构将逆转录病毒载体中的基因传递给人类角质形成细胞。这些含有高比例干细胞的细胞将被移植到FVIII缺陷免疫缺陷小鼠身上，以显示从转基因角质形成细胞中纠正FVIII缺陷。此外，我们的后续观察表明，皮内注射FVIII转基因可以导致FVIII的长期循环。在提案的第二部分，我们试图改进通过尾静脉注射在腺病毒载体中传递的小鼠FVIII基因实现的适度长期校正。我们测试了一些变量，包括改进的基因结构和短期免疫抑制。我们还使用“内脏”的腺病毒载体作为载体来改善结果。这些研究应该提供关于最佳治疗模式的充分信息，以证明在赠款的最后几年纠正血友病A犬模型的尝试是合理的。在第四个特定目标中，我们启动了一种新的载体，内源性L1逆转座子的研究，并确定了这个有希望的载体在培养细胞中传递因子IX基因的有效性。

This proposal sets the stage for clinical trials of gene therapy for hemophilia A, while simultaneously initiating studies on a novel vector system. We initially will determine the most effective and safest gene correction strategy for factor VIII (FVIII) deficiency in our mouse model. We have previously shown that functional VIII can be delivered from the suprabasal epidermis to the circulation in a transgenic mouse and a skin explant. We will improve FVII delivery by use of 1) a FVII resistant to inactivation and 2) a species-specific FVIII. We then use the optimal FVIII construct to deliver the gene in a retroviral vector to human keratinocytes. These cells which contain a high fraction of stem cells will be grafted to FVIII-deficient immunodeficient mice to show correction of FVIII-deficiency from transfected keratinocytes. In addition, we follow- up observations suggesting that intradermal injection of an FVIII transgene can result in long-term circulating FVIII. In a second section of the proposal, we seek to improve upon modest long-term correction achieved by mouse FVIII cDNA delivered in an adenoviral vector by tail vein injection. We test a number of variables, including improved gene construct and short-term immunosuppression. We also use a "gutted" adenoviral vector as a delivery vehicle to improve results. These studies should provide sufficient information concerning optimal modes of therapy to justify attempts to correct a dog model of hemophilia A in the later years of the grant. In a fourth Specific Aim, we initiate studies on a novel vector, the endogenous L1 retro-transposon and determine the efficacy of this promising vector in the delivery of the factor IX gene in cultured cells.