SILENCING RESISTANT GLOBIN RETROVIRAL VECTORS

逆转录病毒介导的具有抗镰状细胞特性的珠蛋白基因衍生物的基因转移到造血干细胞（hsc）中仍然是镰状细胞贫血基因治疗的最有前途的方法之一。我们最近证明，用含有人β -珠蛋白基因顺式连接基因座控制区（LCR）片段和绿色荧光蛋白表达盒的逆转录病毒转导的造血干细胞的离体预选择导致完整的造血所有移植小鼠用转导细胞重建，红细胞长期表达人β -珠蛋白RNA和蛋白。现在很清楚，这些现象是大多数LCR衍生物无法在单拷贝整合的顺链基因上赋予位置无关表达的直接后果。因此，Specific Aims 1和2将研究β -或γ -珠蛋白/LCR逆转录病毒是否可以被修饰以防止基因沉默。根据我们对染色质介导的转录的最新进展，可能的方法包括：(i)利用染色质绝缘体来修饰转移的β -珠蛋白基因；（ii）对珠蛋白/LCR DNA插入中的关键CpG二核苷酸进行位点定向突变，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逆转录病毒结构将在单前病毒复制小鼠转基因和骨髓移植实验中同时进行评估。Specific Aim 3将努力提高我们在不重排的情况下实现高滴度病毒生产这种复杂遗传结构的能力。这需要独家生产全长（未剪接）病毒RNA及其有效包装。将评估两种方法。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乙型肝炎病毒的主要转录稳定剂与HIV-1 Rev/RRE组分结合使用，这是迄今为止最有效的核-细胞质输出机制；（ii）使用来自Semliki Forest病毒（SFV）的RNA载体，其RNA只在细胞质中复制，将逆转录病毒RNA递送到包装细胞中，从而在包装之前消除其在细胞核中的不必要剪接。最后，这些优化珠蛋白/LCR结构及其在高滴度病毒中的传递的策略将在Specific Aim 4中适用于HIV-1衍生的慢病毒载体，以高效、快速地在体外转导静止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.