Development of improved lentiviral vectors for human gene therapy applications

大量证据已经揭示了先天免疫系统的一个可区分的分支，它降低了载体货物进入干细胞的效率。我们假设静止的祖细胞表达先天免疫因子来抵抗病原体，转导进一步介导免疫反应，减少病毒预整合复合体（PIC）的形成，阻止有效的传递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物中的内源性表达，以及那些用VSV-G假型慢病毒载体转导的基因，以将先天基因的表达与转导效率联系起来。CD34+转导培养物中的先天免疫基因反应可能赋予诱导和维持强大的内在抗病毒反应的能力，并随后降低转导效率。因此，通过研究哪些先天因子对转导有反应，我们将针对这些特定因子来抵消它们的免疫活性，以提高转导效率。改善载体传递的潜力将有助于更有效地在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，转基因表达可能会由于甲基化和其他方式的沉默而持续下降。伽玛卫星DNA已经在人类染色体的百分分体区域被鉴定出来。伽玛卫星DNA是一个220bp的CG-reich重复单元串联阵列，通常形成10-200 kb的簇。其功能仍然模糊不清。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γ -卫星DNA将从SIN慢病毒载体中提供稳定的突变基因表达。

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.