Gene correction for Hemophilia A

这一建议为血友病A基因治疗的临床试验奠定了基础，同时启动了一种新的载体系统的研究。我们将首先确定最有效和最安全的因子VIII （FVIII）缺陷的基因校正策略。我们之前已经在转基因小鼠和皮肤外植体中证明功能性VIII可以从基底上表皮传递到循环系统。我们将通过使用1)抗失活的FVII和2)物种特异性FVIII来改善FVII的递送。然后，我们使用最佳的FVIII结构将基因在逆转录病毒载体中传递到人角质形成细胞。这些含有高比例干细胞的细胞将被移植到fviii缺陷免疫缺陷小鼠中，以显示从转染的角化细胞中纠正fviii缺陷。此外，我们的随访观察表明皮内注射FVIII转基因可导致长期循环FVIII。在提案的第二部分中，我们寻求改进通过尾静脉注射在腺病毒载体中传递小鼠FVIII cDNA所实现的适度长期校正。我们测试了许多变量，包括改进的基因构建和短期免疫抑制。我们还使用“去内脏”腺病毒载体作为递送载体来改善结果。这些研究应该提供关于最佳治疗模式的足够信息，以证明在资助的后期试图纠正狗血友病a模型是合理的。在第四个特定目标中，我们开始研究一种新的载体，内源性L1逆转录转座子，并确定这种有前途的载体在培养细胞中传递因子IX基因的功效。

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.