A Novel Genetic Therapy for Herpes Keratitis

疱疹性角膜炎的新型基因疗法

• 批准号: 7123811
• 负责人: JOHN D KRIESEL
• 金额: $ 14.6万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7123811
• 关键词: biotechnology; chemical association; gene delivery system; gene therapy; genetic regulation; genetic transcription; herpes simplex virus 1; inhibitor /antagonist; injection /infusion; keratitis; laboratory mouse; laboratory rat; latent virus infection; nervous system disorder therapy; neurons; newborn animals; nonhuman therapy evaluation; oligonucleotides; relapse /recurrence; retinal ganglion; trigeminal nerve; virus DNA; virus protein; virus replication

DESCRIPTION (provided by applicant): This proposal seeks to develop triplex oligonucleotide (known as "triplex oligo", "TFO", or "antigene") technology into a useful therapy for herpes keratitis. Triplex oligo is based on the observation that a third DNA strand - the triplex - can bind non-covalently to specific double-stranded DNA target sequences to induce mutation or affect transcription. We have identified several such target sequences within the HSV-1 genome, including two in the latency-associated transcript (LAT) region. Targeting LAT, a crucial part of the HSV genome, is proposed as a logical first step toward the prevention of HSV-1 gene transcription and, hence, ocular reactivation. This novel genetic therapy could potentially prevent thousands of episodes of recurrent herpes keratitis each year. A large amount of preliminary data is presented to support the feasibility of the proposed studies. The specific aims of the proposal are: 1. Mechanism of action of photoactivatable TFO's. Control pheophorbide conjugated TFO's matched to TFO-1 and TFO-3 in length and AG composition will be sythesized. TFO-1-pheo and TFO-3-pheo alone and in combination will be retested using the appropriate control oligonucleotides. The mechanism of antiviral activity of the pheophorbide-linked TFO's will be investigated by specific cleavage site determinations, guanine oxidation, and cross-linking experiments. 2. Delivery of anti-HSV-1 Triplex-Forming Oligonucleotides. Various strategies for delivery of anti-HSV-1 TFO's into the TG's of live mice will be examined with an emphasis on intraperitoneal and/or intravenous infusion of oligonucleotides. The integrity of the delivered oligos will be determined by native gel electrophoresis of TG cellular extracts. 3. Prevention of HSV-1 Ocular Reactivation. The mouse model of HSV-1 ocular infection and reactivation will be used to investigate whether triplex oligos can inhibit HSV-1 induced ocular reactivation. An ex vivo model of HSV-1 reactivation in explanted mouse trigeminal ganglia is proposed as an efficient means for determining effective strategies for the delay or inhibition of reactivation.

描述（由申请人提供）：该提案旨在开发三核苷酸（称为“ Triplex Oligo”，“ TFO”或“抗基因”）技术，成为用于疱疹角膜炎的有用疗法。 Triplex Oligo基于以下观察结果：第三个DNA链（三链）可以非共价结合特定的双链DNA靶序列以诱导突变或影响转录。我们已经确定了HSV-1基因组中的几个此类目标序列，包括与潜伏期相关的转录本（LAT）区域中的两个。靶向LAT是HSV基因组的关键部分，被认为是迈向预防HSV-1基因转录的逻辑第一步，因此是眼重新激活。这种新型的基因疗法可能有可能预防每年数千次复发性疱疹发作。提供了大量初步数据，以支持拟议研究的可行性。 该提案的具体目的是： 1。可光活化TFO的作用机理。控制质磷酸的结合TFO将与TFO-1和TFO-3的长度和Ag组成相匹配。仅使用适当的对照寡核苷酸来重新测试TFO-1-Pheo和TFO-3-Pheo和组合。通过特定的切割部位的测定，鸟嘌呤氧化和交联实验，将研究质质膜相关的TFO的抗病毒活性的机理。 2。抗HSV-1形成三核苷酸的抗HSV-1。将检查将抗HSV-1 TFO输送到活小鼠TG的各种策略，重点是腹膜内和/或静脉输注寡核苷酸。传递的寡核体的完整性将由TG细胞提取物的天然凝胶电泳确定。 3。预防HSV-1眼睛重新激活。 HSV-1眼部感染和重新激活的小鼠模型将用于研究三元寡核能是否可以抑制HSV-1诱导的眼反性激活。在外植的小鼠三叉神经节中，HSV-1重新激活的离体模型被认为是确定延迟或抑制重新激活的有效策略的有效手段。