Targeted Disruption of Integrated SHIV by Engineered Homing Endonucleases

工程化归巢核酸内切酶有针对性地破坏整合的 SHIV

• 批准号: 8202339
• 负责人: KEITH R JEROME
• 金额: $ 58.68万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-08 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8202339
• 关键词: Autologous; Base Pairing; Biochemical; Biological Assay; CD34 gene; CD4 Positive T Lymphocytes; Cells; Cessation of life; Clinical; Coculture Techniques; Collaborations; Computer Assisted; Computer Simulation; Conserved Sequence; Coupled; DNA; DNA Binding; DNA Double Strand Break; Disease; Engineering; Ensure; Enzymes; Family; Frequencies; Generations; Genetic; Genetic Materials; Genome; Goals; HIV; HIV Infections; Hematopoietic Stem Cell Transplantation; Homing; Human; Immune; Immune system; Incidence; Individual; Laboratories; Lead; Life; Link; Macaca; Mammalian Cell; Measurable; Mediating; Methods; Modeling; Mutagenesis; Mutate; Mutation; Nonhomologous DNA End Joining; Pathogenesis; Patients; Primates; Process; Proteins; Proviruses; Reporter; Research Personnel; Screening procedure; Site; Site-Directed Mutagenesis; Solutions; Specificity; Stem cells; Suggestion; System; T memory cell; TREX2 gene; Testing; Time; Variant; Vesicular stomatitis Indiana virus; Viral; Viral Load result; Virus; base; design; directed evolution; ds-DNA; endonuclease; exodeoxyribonuclease; immune function; improved; in vivo; innovation; latent infection; member; novel; novel strategies; prevent; purge; reconstitution; repaired; simian human immunodeficiency virus; success

Current treatments do not cure individuals of HIV because of HIV's ability to persist as provirus within long-lived immune cells, especially memory T cells. Most attempts to clear these reservoirs have involved the destruction of latently infected cells, and such efforts to date have been unsuccessful. A more elegant solution to the problem of HIV infection would be the ability to eliminate or otherwise disable integrated HIV without inducing death of infected cells. We have recently begun to evaluate a novel approach to achieve this, via the use of novel, highly specific enzymes known as homing endonucleases (HEs). HEs are proteins that specifically target long (~20 base pair) sequences in double stranded DNA. The long recognition sites ensure high specificity and prevent off-target effects. Upon recognition of their target sites, HEs induce DNA double strand breaks (dsb) at the sites, which in mammalian cells are most commonly repaired via non-homologous end joining (NHEJ). NHEJ repair is error prone, and can lead to mutation typically resulting in deletions surrounding the cleavage site ranging from 1 base to several kilobases. In our preliminary studies, we created an engineered reporter virus containing the recognition site for the natural HE l-Anil, to demonstrate that integrated provirus can be successfully targeted for attack, cleavage, and mutagenesis by HEs. Based on this success, we have now assembled a team of investigators with the complementary expertise necessary to rationally redesign natural homing endonucleases such that they will recognize conserved sequences within SHIV. Our aims are as follows. 1) Design variant homing endonucleases targeting integrated SHIV sequences. We will build upon existing collaborations with Drs. David Baker and Andrew Scharenberg, with whom we have already used computational and bacterial selection approaches to design novel HEs recognizing conserved sequences from HIV. 2) Optimize the DNA-modifying efficacy of SHIV-specific homing endonucleases. We will do this by directed evolution processes for selection of increased activity, and by linking the HEs to TREX2, which we have shown increases the incidence of targeted mutation. 3) Establish the optimal methods for delivery of SHIV-specific HEs to macaque CD4+ T cells and CD34+ stem cells, and determine the ability of HEs to clear viable provirus from these reservoirs. During autologous hematopoietic stem cell transplantation in HIV-infected individuals, between 10[5] and 10[10] copies of HIV provirus are re-infused along with the graft, and the ability to purge viable virus from these cells before reintroduction may be of clinical benefit to the patient.

目前的治疗方法不能治愈艾滋病毒的个人，因为艾滋病毒的能力，坚持作为前病毒在长期生存， 免疫细胞，尤其是记忆T细胞。大多数清除这些水库的尝试都涉及到 破坏潜伏感染的细胞，并且迄今为止这种努力没有成功。一个更优雅 解决艾滋病毒感染问题的办法是有能力消除或以其他方式使综合的艾滋病毒丧失能力 而不诱导受感染细胞的死亡。我们最近开始评估一种新的方法， 这是通过使用称为归巢核酸内切酶（HEs）的新型高度特异性的酶实现的。HE是蛋白质 其特异性靶向双链DNA中的长（~20个碱基对）序列。长识别位点 确保高特异性并防止脱靶效应。在识别其靶位点后，HE诱导DNA 在哺乳动物细胞中最常见的是通过非同源的DNA修复的位点处的双链断裂（dsb）。 端部连接（NHEJ）。NHEJ修复是容易出错的，并且可以导致突变，通常导致 切割位点周围的缺失，范围从1个碱基到几个碱基。在我们的初步研究中， 我们创造了一种工程报道病毒，它含有天然HEl-Anil的识别位点， 证明整合的前病毒可以成功地靶向攻击、切割和诱变， 他。基于这一成功，我们现在已经组建了一个调查小组， 合理地重新设计天然归巢核酸内切酶所需的专业知识， SHIV中的保守序列。我们的目标如下。1)设计变体归巢核酸内切酶 靶向整合的SHIV序列。我们将在现有的合作基础上与大卫贝克博士和 Andrew Scharenberg，我们已经与他一起使用了计算和细菌选择方法， 设计识别HIV保守序列的新型HE。2)优化DNA修饰功效 SHIV特异性归巢核酸内切酶。我们将通过定向进化过程来选择 增加的活性，并通过连接HEs与TREX 2，我们已经表明增加的发病率 定向突变3)建立向猕猴CD 4 + T细胞递送SHIV特异性HE的最佳方法 细胞和CD 34+干细胞，并确定HE从这些储库中清除活前病毒的能力。 在HIV感染者的自体造血干细胞移植期间，10[5]至10[10] HIV前病毒的拷贝与移植物一起沿着被重新注入， 在再引入之前的细胞可能对患者具有临床益处。