SILENCING RESISTANT GLOBIN RETROVIRAL VECTORS

逆转录病毒介导的抗镰状化珠蛋白基因转移到造血干细胞（HSC）中是镰状细胞性贫血基因治疗的最有前途的方法之一。我们最近证明，用含有与基因座控制区（LCR）片段顺式连接的人β-珠蛋白基因的逆转录病毒转导HSC，而绿色荧光蛋白表达盒导致所有移植小鼠用转导细胞的完全造血重建以及人β-珠蛋白RNA和蛋白质在红细胞中的长期表达。现在清楚的是，这些现象是大多数LCR衍生物不能赋予以单拷贝整合的顺式连接的基因位置非依赖性表达的直接结果。因此，特定目的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。基于我们对染色质介导的转录的理解的最新进展，可能的方法将包括：（i）利用染色质绝缘体侧接转移的β-珠蛋白基因和（ii）珠蛋白/LCR DNA插入物中的关键CpG二核苷酸的定点诱变，以使其不能被甲基化机制检测到。这些不可表达的珠蛋白/LCR DNA插入使其不能被甲基化机制检测到。将在单原病毒拷贝小鼠转基因和骨髓移植实验中同时评价这些非阻遏性珠蛋白/LCR逆转录病毒构建体。具体目标3将努力提高我们的能力，实现高滴度病毒生产的这种复杂的遗传结构没有重排。这需要专门生产全长（未剪接）病毒RNA及其有效包装。将对两种方法进行评价。这需要专门生产全长（未剪接）病毒RNA及其有效包装。将评价两种方法：（i）掺入来自伍德查克B肝炎病毒的初级转录物稳定剂与HIV-1 Rev/RRE组分的组合，这是迄今为止表征的最有效的核质输出机制，和（ii）使用来自塞姆利基森林病毒（SFV）的RNA载体，其RNA仅在细胞质中复制，以将逆转录病毒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需要的剪接。最后，这些用于优化珠蛋白/LCR构建体的结构及其在高滴度病毒中的递送的策略将在特异性目的4中适用于HIV-1衍生的慢病毒载体，以有效和快速地离体转导静止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.