PATRICK LEE GREEN (2019-02-13 to 2021-01-31) Genome editing to prevent HTLV-1 disease. Amount: $195000

基因组编辑，以防止HTLV-1疾病

Abstract

Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus and the causative infectious agent of both HTLV-1- associated myelopathy/tropical spastic paraparesis (HAM/TSP), a chronic inflammatory disease of the central nervous system, and adult T-cell leukemia/lymphoma (ATL), an aggressive and fatal disease of CD4+ T cells. An ideal therapeutic strategy against HAM/TSP is still not established and once neurological symptoms begin, they progress quickly leading to deterioration in quality of life. ATL is chemotherapy-resistant with a median survival time of <1 year. The lack of effective therapies suggests an imminent need for new approaches to treatment or prevention. We propose a novel approach utilizing the CRISPR bacterial immunity system to disable or disrupt the HTLV-1 proviral genome and cell proliferative and ultimately tumorigenic effect on T cells. The CRISPR system includes the Cas9 endonuclease that is targeted by a guide RNA (gRNA) to introduce a specific double strand break (DSB) into genomic DNA. These DSBs are largely repaired by the error-prone non-homologous end- joining (NHEJ) pathway in most human cells, which typically introduces insertions and deletions (indels) at the repair junction. Indels may alter the reading frame of genes or disrupt essential regulatory elements or RNA stem-loop structures. However, the broad effects of using the CRISPR system to target the HTLV-1 genome are unknown. This proposal contains two highly focused Specific Aims: 1.) Utilize CRISPR/Cas9 technology to disable the HTLV-1 provirus and its proliferative inducing and tumorigenic effects on infected T cells, and 2.) To determine the therapeutic efficacy of CRISPR/Cas9-based genome editing against HTLV-1 NOG SCID transplant mouse model. We will generate a library of CRISPR gRNAs targeting key regions of the HTLV-1 genome, including the long terminal repeats (LTRs), the tax gene, and the hbz gene. This library will be characterized for viral infectivity, transcription, cellular proliferation, and off-target editing. The nature and location of mutations that effectively disable HTLV-1 will be analyzed by sequencing. We will further determine if the CRISPR induced mutations prevent HTLV-1 cell growth and tumor induction in vivo. Ultimately, this exploratory grant will provide a quantitative and adaptable platform for probing the efficiency of CRISPR editing to disable HTLV-1 and prevent or disrupt viral gene expression, infected cell growth and ultimately host neurological/immunological complications or oncogenesis. These data will yield significant insights into the suitability and practicality of genome editing technologies targeting HTLV-1.

人类T细胞白血病病毒1型（HTLV-1）是一种逆转录病毒，是HTLV-1-相关性脊髓病/热带痉挛性下肢瘫痪（HAM / TSP）的致病感染因子，是中枢神经系统的慢性炎症性疾病，成人T细胞白血病/淋巴瘤（ATL），CD4 + T细胞的侵袭性和致命性疾病。尚未确定针对HAM / TSP的理想治疗策略，并且一旦神经症状开始，它们就会迅速发展，导致生活质量下降。 ATL具有化疗耐药性，中位生存期<1年。缺乏有效的治疗方法表明迫切需要新的治疗或预防方法。我们提出了一种新方法，利用CRISPR细菌免疫系统来禁用或破坏HTLV-1原病毒基因组和细胞增殖以及最终对T细胞的致瘤作用。 CRISPR系统包括Cas9内切核酸酶，其被指导RNA（gRNA）靶向以将特定的双链断裂（DSB）引入基因组DNA中。这些DSB在很大程度上通过大多数人类细胞中容易出错的非同源末端连接（NHEJ）途径进行修复，这通常在修复连接处引入插入和缺失（插入缺失）。 Indel可能改变基因的阅读框架或破坏必需的调节元件或RNA茎环结构。然而，使用CRISPR系统靶向HTLV-1基因组的广泛影响尚不清楚。该提议包含两个高度集中的特定目标：1。利用CRISPR / Cas9技术禁用HTLV-1原病毒及其对受感染T细胞的增殖诱导和致瘤作用，以及2.）确定CRISPR / Cas9-的治疗功效基于HTLV-1 NOG SCID移植小鼠模型的基因组编辑。我们将生成靶向HTLV-1基因组关键区域的CRISPR gRNA文库，包括长末端重复序列（LTRs），tax基因和hbz基因。该文库将表征病毒感染性，转录，细胞增殖和脱靶编辑。将通过测序分析有效抑制HTLV-1的突变的性质和位置。我们将进一步确定CRISPR诱导的突变是否阻止体内HTLV-1细胞生长和肿瘤诱导。最终，这一探索性资助将提供一个定量和适应性平台，用于探测CRISPR编辑的效率，以禁用HTLV-1并预防或破坏病毒基因表达，感染细胞生长并最终宿主神经/免疫并发症或肿瘤发生。这些数据将为针对HTLV-1的基因组编辑技术的适用性和实用性提供重要的见解。

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