Developing novel deep sequencing techniques for studying HIV evolution

开发用于研究 HIV 进化的新型深度测序技术

• 批准号: 7930481
• 负责人: Stephanie Michelle Willerth
• 金额: $ 1.3万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-27 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7930481
• 关键词: Aftercare; Alternative Therapies; Antibiotic Resistance; Antisense RNA; Bacteria; Clinical; Collaborations; Computer Simulation; DNA Sequence; Development; Evolution; Future; Genetic Models; Genome; HIV; Highly Active Antiretroviral Therapy; Influenza; Lentivirus Vector; Methods; Mutate; Population; Population Genetics; Resistance; Resistance development; Sampling; Techniques; Therapeutic Intervention; Time; Vesnarinone; Viral; Virus; West Nile virus; Work; base; data modeling; design; effective therapy; insight; next generation; novel; pathogen; pressure; public health relevance; response

DESCRIPTION (provided by applicant): One of the major obstacles to developing suitable therapies for the treatment of specific pathogens is their ability to mutate and evolve in response to therapeutic intervention (1), including the emergence of antibiotic- resistant bacteria, yearly strains of influenza, and human immunodeficiency virus (HIV) strains that become resistant to the effects of highly active antiretroviral therapy (HAART). With 38 million people infected with HIV worldwide, it is important to understand how this virus mutates and develops resistance in order to design successful therapies. The central objective of this proposal is to analyze how populations of HIV mutate over time in response to evolutionary pressure, in this case an RNA antisense therapy. This study will use novel "next generation" DNA sequencing methods because they may generate near-complete coverage of the HIV genomes present in clinical samples. Specifically, I will sequence the HIV quasispecies population both before and after treatment with the lentiviral vector VRX496", which encodes an antisense sequence that targets the envelope of HIV, allowing me to study the impact of therapeutic intervention on HIV evolution over time. Furthermore, the results will enable me to develop a computational, population genetics model of quasispecies evolution over time. The resulting data and model may in the future aid the design of more effective treatments for people infected with HIV. The experimental portion of this proposal will be performed in the lab of Dr. David Schaffer (sponsor) with collaboration from Dr. Arkin's lab for computational modeling, and clinical samples used for analysis will be obtained from the VIRxSYS corporation. Aim 1. To determine whether the development of an unbiased sequencing method can yield >10,000x coverage of the HIV genome. Aim 2. To determine whether next generation sequencing can be applied to elucidate mechanisms of HIV evolution in response to a selective pressure - specifically a lentiviral-based antisense therapy. Aim 3. To determine if an accurate computational model of HIV quasispecies evolution in response to a selective pressure can be developed. PUBLIC HEALTH RELEVANCE: An estimated 38 million people worldwide are currently infected with HIV with an additional 4.1 million people becoming infected each year. There is a compelling need to develop alternative therapies for the treatment of HIV due to the limitations of HAART, and this work will yield deep insights into viral responses to a new lentiviral-based antisense therapy. Additionally, the general methods described in this work will be applicable to studying other highly heterogeneous, rapidly evolving viral populations, such as influenza, West Nile virus, Ebola, and others.

描述（由申请人提供）：开发用于治疗特定病原体的合适疗法的主要障碍之一是它们响应治疗干预而突变和进化的能力（1），包括出现抗生素耐药细菌、流感年度株和对高效抗逆转录病毒疗法（HAART）产生耐药性的人类免疫缺陷病毒（HIV）株。全世界有3800万人感染艾滋病毒，了解这种病毒如何变异和产生耐药性，以便设计成功的疗法，这一点很重要。这项提案的中心目标是分析艾滋病毒群体如何随着时间的推移而突变，以应对进化压力，在这种情况下，RNA反义疗法。这项研究将使用新的“下一代”DNA测序方法，因为它们可能会产生几乎完全覆盖临床样本中存在的HIV基因组。具体来说，我将在用慢病毒载体VRX 496”治疗之前和之后对HIV准种群体进行测序，该载体编码针对HIV包膜的反义序列，使我能够研究治疗干预对HIV演变的影响随着时间的推移。此外，这些结果将使我能够开发一个计算，随着时间的推移准物种进化的群体遗传学模型。由此产生的数据和模型可能在未来有助于为艾滋病毒感染者设计更有效的治疗方法。本提案的实验部分将在Dr.大卫·谢弗（申办方）的实验室中进行，并与Dr. Arkin的实验室合作进行计算建模，用于分析的临床样本将从VIRxtek公司获得。目标1.确定无偏测序方法的开发是否可以产生>10，000 x的HIV基因组覆盖率。目标2.确定下一代测序是否可用于阐明HIV进化机制，以响应选择性压力-特别是基于慢病毒的反义治疗。目标3.确定是否可以开发一个精确的HIV准种进化响应选择压力的计算模型。 公共卫生相关性：据估计，全世界目前有3 800万人感染艾滋病毒，每年还有410万人感染。由于HAART的局限性，迫切需要开发用于治疗HIV的替代疗法，这项工作将深入了解病毒对新的基于慢病毒的反义疗法的反应。此外，这项工作中描述的一般方法将适用于研究其他高度异质性，快速进化的病毒种群，如流感，西尼罗河病毒，埃博拉病毒等。