Nonintegrating Lentiviral Vectors Towards Clinical Trials

 描述：我们实验室最近发表了一份报告，证明了整合有缺陷的慢病毒载体(IDLV)在临床前环境中治愈血友病B的能力，为其治疗潜力提供了进一步的证据。然而，需要进一步改进IDLV基因传递系统，以建立IDLV作为治疗诸如血友病B等非致命性人类疾病的方式。因此，我们建议：a)使用新的PPT缺失载体来进一步降低与系统给药IDLV相关的插入突变的低风险，b)开发和测试新的基于IDLV的人凝血因子IX(HFIX)表达盒作为一种手段，以最小化完全纠正血友病B小鼠缺陷所需的IDLV载体载量，以及c)建立一种新的包装细胞系，以便于生产动员抗性IDLV的携带表达框，其方向与载体的LTR相反。在目标1中，我们将重点研究PPT缺失、非法整合减少的新型IDLV在体内介导有效的肝脏基因传递的能力。目的2将致力于开发和测试一种新的hFIX基因，该基因具有较长的体内半衰期(T1/2)和增强的比活性，可用于肝脏基因传递。将在血友病B小鼠身上测试新的IDLV纠正Fix缺陷的有效性。目标3的重点将是建立一种新型的RNA调节蛋白激酶(PKR)抗性包装细胞系，产生高滴度的gp64假型载体。PPT缺失将使细胞系产生的IDLV不太可能非法整合，并且内部表达盒以与LTR意愿相反的方向结合 使他们不太可能被动员起来。总体而言，拟议的研究将产生一个高效的 以及更安全的基因传递系统，最适合于非致命性人类疾病的基因替代治疗。

 DESCRIPTION: Our laboratory recently published a report demonstrating the ability of integration defective lentiviral vectors (IDLV's) to cure hemophilia B in preclinical settings, providing additional proof of their therapeutic potential. However, further improvements of the IDLV gene delivery system are required in order to establish IDLV's as a therapeutic modality for nonfatal human diseases such as hemophilia B. Thus, we propose: a) to employ a novel PPT-deleted vector to further reduce the low risk of insertional mutagenesis associated with systemic administration of IDLV's, b) to develop and test a novel IDLV-based human factor IX (hFIX) expression cassette as a means to minimize IDLV vector load required to fully correct FIX deficiency in hemophilia B mice, and c) to establish a novel packaging cell line to facilitate production of mobilization resistant IDLV's carrying expression cassettes in opposite orientation to the vector's LTR's. The proposal comprises three specific aims. In Aim 1 we will focus on characterizing the ability of novel PPT-deleted IDLV's with reduced illegitimate integration to mediate efficient hepatic gene delivery, in vivo. Aim 2 will focus on the development and testing of a novel hFIX cDNA with prolonged in vivo half-life (t1/2) and enhanced specific activity using IDLV's for hepatic gene delivery. The efficacy of the new IDLV's to correct FIX deficiency will be tested in hemophilia B mice. The focus of Aim 3 will be establishing of a novel RNA-regulated protein kinase (PKR) resistant packaging cell line generating high titers of gp64- pseudotyped vectors. A PPT-deletion will render the cell line-generated IDLV's less likely to illegitimately integrate, and internal expression cassettes incorporated in opposite orientation to the LTR's will render them less likely to be mobilized. Overall the proposed studies will yield a highly efficient and significantly safer gene delivery system most suitable for gene replacement therapy of nonfatal human diseases.