SILENCING RESISTANT GLOBIN RETROVIRAL VECTORS

逆转录病毒介导的具有抗镰刀特性的珠蛋白基因衍生物基因转移到造血干细胞(HSCs)仍然是治疗镰状细胞性贫血的最有前途的方法之一。我们最近证明，体外预选转导HSCs的逆转录病毒含有顺式连接的人β-珠蛋白基因位点控制区(LCR)片段和绿色荧光蛋白表达盒，可使所有移植的小鼠完全重建造血系统，并在红细胞中长期表达人β-珠蛋白RNA和蛋白。现在很清楚，这些现象是大多数LCR衍生物不能在单拷贝整合的顺式连接基因上进行位置无关表达的直接后果。因此，特定的AIMS 1和2将调查是否可以修改β或伽马珠蛋白/LCR逆转录病毒以防止基因沉默。根据我们对染色质介导的转录调控的最新进展，可能的方法包括：(I)利用染色质绝缘体来侧翼转移的β-珠蛋白基因；(Ii)对珠蛋白/LCRDNA插入片段中的关键CpG二核苷酸进行定点突变，使其无法被甲基化机制检测到。这些不可表达的珠蛋白/LCR DNA插入，使其无法被甲基化机制检测到。这些不可抑制的珠蛋白/LCR逆转录病毒构建物将在单一前病毒复制小鼠转基因实验和骨髓移植实验中同时进行评估。具体目标3将努力提高我们在不重排的情况下实现这种复杂遗传结构的高滴度病毒生产的能力。这需要独家生产全长(未剪接)病毒RNA及其有效包装。将对两种方法进行评估。这需要独家生产全长(未剪接)病毒RNA及其有效包装。将评估两种方法：(I)将Wood Chuck乙肝病毒的初级转录稳定剂与HIV-1Rev/RRE组分结合起来，这是迄今最有效的核质输出机制；(Ii)使用来自Semliki森林病毒(SFV)的RNA载体，其RNA只在细胞质中复制，从而将逆转录病毒RNA输送到包装细胞中，从而在包装之前消除其在细胞核中不必要的剪接。最后，这些优化珠蛋白/LCR结构及其在高滴度病毒中传递的策略将被用于针对HIV-1来源的慢病毒载体，以高效和快速地体外转导静止的HSC。

Retrovirus-mediated gene transfer of a globin gene derivative with anti- sickling properties into hematopoietic sem cells (HSCs) remains one of the most promising approaches for the gene therapy of sickle cell anemia We have recently demonstrated that ex-vivo pre-selection of HSCs transduced with a retrovirus containing the human beta-globin gene cis- linked to fragments of the Locus Control Region (LCR) and a Green Fluorescent Protein expression cassette results in complete hematopoietic reconstitution of all transplanted mice with transduced cells and long- term expression of human beta-globin RNA and protein in red blood cells. It is now clear that these phenomena are the direct consequence of the inability of most LCR derivatives to confer position-independent expression on a cis-linked gene integrated at single copy. Accordingly, Specific Aims 1 and 2 will investigate whether or beta- or gamma- globin/LCR retroviruses can be modified to prevent gene silencing. Based on recent advances in our understanding of chromatin-mediated transcriptional possible approaches will include: (i) utilization of chromatin insulators to flank the transferred beta-globin gene and (ii) site- directed mutagenesis of critical CpG dinucleotides in the globin/LCR DNA insert to make it undetectable by the methylation machinery. These non-expressible globin/LCR DNA insert to make it undetectable by the methylation machinery. These non-repressible globin/LCR retroviral constructs will be evaluated concurrently in single proviral copy mouse transgenics and in bone marrow transplantation experiments. Specific Aim 3 will strive to enhance our ability to achieve high-titer viral production of such complex genetic structures without rearrangement. This requires the exclusive production of full-length (unspliced) viral RNA and its efficient packaging. Two approaches will be evaluated. This requires the exclusive production of full-length (unspliced) viral RNA and its efficient packaging. Two approaches will be evaluated: (i) incorporation of a primary transcript stabilizers from the Wood Chuck Hepatitis B virus combined with HIV-1 Rev/RRE components, the most efficient nucleo-cytoplasmic export machinery characterized to date, and (ii) use of an RNA vector derived from the Semliki Forest virus (SFV), whose RNA replicates exclusively in the cytoplasm, to deliver the retroviral RNA into packaging cells and thereby eliminate its unwanted splicing in the nucleus prior to packaging. Finally, these strategies for optimizing both the structure of the globin/LCR construct and its delivery in high titer virus will be adapted in Specific Aim 4 to HIV-1- derived lentiviral vectors for efficient and rapid ex-vivo transduction of quiescent HSC.