Development of improved lentiviral vectors for human gene therapy applications

大量证据表明，先天免疫系统有一个明显的分支，它降低了将载体货物有效地运送到干细胞的能力。我们假设静止的祖细胞表达先天免疫因子来抵御病原体，转导进一步介导免疫反应，减少病毒前整合复合体(PIC)的形成，阻止有效的递送和整合到宿主染色质中。我们目前正在分析先天免疫基因在造血祖细胞培养和VSV-G伪型慢病毒载体转导的造血祖细胞中的内源性表达，以期将先天免疫基因的表达与转导效率联系起来。在转导CD34+的培养物中，先天免疫基因的反应性可能赋予诱导和维持强大的内在抗病毒反应的能力，从而降低转导效率。因此，通过研究哪些先天因素对转导有反应，我们将针对这些特定的因素来抵消它们的免疫活性，以提高转导效率。改进载体传递的潜力将有助于更有效地临床使用病毒载体作为基因治疗的工具。在成功地将基因转移到造血干细胞和祖细胞后，由于甲基化和其他方式的沉默，转基因表达可能会超时下降。已在人类染色体的百分体区域中发现了伽马卫星DNA。伽马卫星DNA是一个由220bp的CG-Reich重复单元组成的串联阵列，通常形成10-200kb的簇。这一功能仍然模糊不清。然而，美国国立卫生研究院研究人员最近的一项工作表明，重复的DNA可能具有绝缘体活性。我们假设伽马卫星DNA将提供来自SIN慢病毒载体的稳定的trangene表达。 我们克隆了不同大小的伽马卫星重复DNA片段，并将这些片段插入到基于HIV-1的慢病毒载体的3‘LTR缺失点，以评价这些片段的绝缘子活性。在MSCV启动子的控制下，构建了含有EGFP基因的载体载体：简单对照、插入对照鸡HS4绝缘子和插入不同大小的伽马卫星DNA。用这些慢病毒载体转导人红系细胞，并将长期跟踪载体的表达以监测转基因的表达。 为了在临床前的非人类灵长类动物模型中测试这些慢病毒载体，已经对基于HIV-1的慢病毒载体进行了几次修改，以实现恒河猴来源的造血干细胞的有效转导。这些修改包括使用巨噬细胞嗜性株的亲环素结合域以及猴免疫缺陷病毒衣壳的部分。对这些构建体的测试表明，转导效率有所提高，这些病毒载体现在将在非人类灵长类动物模型中进行测试。

Pools of evidence have revealed a distinguishable arm of the innate immune system that reduces the efficient delivery of vector cargo into stem cells. We hypothesize that quiescent progenitor cells express innate immune factors to protect against pathogens and transduction further mediates an immune response that reduces viral pre-integration complex (PIC) formation and prevents efficient delivery and integration into host chromatin. We are currently analyzing the endogenous expression of innate immune genes in hematopoietic progenitor cultures and those transduced with VSV-G psuedotyped lentiviral vectors to correlate expression of innate genes with transduction efficiency. Innate immune gene responsiveness in CD34+ transduced cultures may confer the ability to induce and maintain a strong intrinsic antiviral response and subsequently decreasing transduction efficiency. So, by investigating which innate factors are responsive to transduction, we will target these specific factors to counteract there immune activity in order to increase transduction efficiency. The potential for improving vector delivery would contribute to more efficient clinical usage of viral vectors as tools for gene therapy. Following successful gene transfer to hematopoietic stem and progenitor cells,transgene expression may fall overtime resulting from silencing through methylation and other means. Gamma-satellite DNA has been identified in the percentromeric regions of human chromosomes. The gamma-satellite DNA is a tandem array of 220bp CG-reich repetitive units, usually forming 10-200 kb clusters. The function remains obscure. However, a recent work by NIH investigators suggests that the repetitive DNA might possess insulator activity. We hypothesize that the gamma-satellite DNA will provide stable trangene expression from SIN lentivirus vectors. We have cloned various sizes of the gamma-satellite repetitive DNA fragments and inserted these fragments into the deletion site of 3'LTR of an HIV-1 based lentiviral vector to evaluate the insulator activity with these fragments. The vector plasmids were constructed containing EGFP cDNA under the control of MSCV promoter: simple control, insertion of the control chicken HS4 insulator and insertion of several sizes of the gamma-satellite DNA. Human erythroid cell lines were transduced with these lentivirus vectors, and vector expression will be followed long term to monitor transgene expression. In order to test these lentiviral vectors in the preclinical nonhuman primate model, several modifications of HIV-1 based lentiviral vectors have been made to enable efficient transduction of rhesus macaque derived hematopoietic stem cells. These modifications include the use of the cyclophillin binding domain of the macrophage tropic strain along with portions of the simian immunodeficiency virus capsid. Testing of these constructs demonstrates improved transduction rates and these viral vectors will now be tested in the nonhuman primate model.