TARGETED CPG OF CCR5 GENE--NOVEL HIV THERAPY

CCR5基因编码嗜巨噬细胞HIV-1毒株的共受体；在缺乏CCR5的情况下，HIV-1感染被阻断。我们建议通过靶向灭活巨噬细胞祖细胞中的CCR5基因来使个体对HIV-1产生抗性。我们描述了一种开发靶向甲基转移酶的方法，该方法将对CCR5基因表达进行强而持久的抑制。DNA甲基化导致转座因子和X失活或基因组印记基因的转录抑制。甲基化模式在S期与DNA一起复制。序列特异性甲基化剂的短暂存在将使靶基因持久失活。我们将开发CCR5启动子特异性DNA甲基转移酶，通过将cpg特异性DNA甲基转移酶与锌指蛋白融合，设计用于结合CCR5基因的近端启动子。该过程包括合理设计和选择新型锌指蛋白。该项目的最终目标是开发一种重组腺相关病毒（AAV），该病毒可以传递靶向甲基转移酶，以选择性地灭活靶细胞群中的CCR5基因。这里开发的策略将在其他疾病治疗中得到广泛应用。拟议的商业应用：CCR5基因的靶向甲基化将使个体对HIV感染具有抵抗力。这将对艾滋病毒治疗和预防感染产生直接影响。这项研究更广泛的潜在应用是选择性地抑制与人类疾病有关的任何人类或病毒基因，从而创造一类新的治疗剂。

The CCR5 gene encodes a co-receptor for macrophage-tropic strains of HIV-1; in the absence of CCR5, HIV-1 infection is blocked. We propose to render individuals resistant to HIV-1 by targeted inactivation of the CCR5 gene in the progenitor cells of macrophages. We describe a method for the development of a targeted methyltransferase that will give strong and enduring inhibition of CCR5 gene expression. DNA methylation is responsible for the transcriptional repression of transposable elements and genes subject to X inactivation or genomic imprinting. Methylation patterns are replicated along with the DNA during S phase. Transient presence of a sequence specific methylating agent will give enduring inactivation of the target gene. We will develop DNA methyltransferases specific to the CCR5 promoter by fusion of CpG-specific DNA methyltransferases to zinc-finger proteins designed to bind to the proximal promoter of the CCR5 gene. The procedure involves rational design and selection of novel zinc finger proteins. The ultimate goal of the project is the development of a recombinant adeno- associated virus (AAV) that can deliver a targeted methyltransferase to selectively inactivate the CCR5 gene in the target cell populations. The strategy developed here will find broad application in other disease therapies. PROPOSED COMMERCIAL APPLICATION: Targeted methylation of the CCR5 gene will render individuals resistant to HIV infection. This will have an immediate impact on HIV therapy and the prevention of infection. The broader potential application of this research is to selectively repress any human or viral gene implicated in human disease, thus creating a new class of therapeutic agents.