Electrochemical RT Activity Assay for Measuring HIV Load

描述(申请人提供)：为了解决在资源有限的情况下对艾滋病毒感染者进行更好的治疗和诊断测试的迫切需要，我们在此第二阶段SBIR中建议进一步开发由Alderon Biosciences，Inc.开发的用于检测临床样本中艾滋病毒病毒载量的电化学测试系统(ESystem)。Alderon的长期目标是生产、验证和获得FDA的批准，使用一种简单、负担得起(10美元)的HIV病毒负载测试，对低于500拷贝/毫升敏感，使用廉价和便携的仪器提供当天的结果。目前可用的FDA批准的HIV病毒载量测试需要昂贵的仪器，使用复杂的分子生物学程序，每次测试可能需要100美元。我们的假设是，Alderon可以开发一种更方便、更实惠的系统，用于准确的病毒载量测量和药物敏感性测试，使病毒载量测试能够在资源有限的护理点(POC)环境中进行。Alderon提出的新的HIV病毒载量检测方法的创新之处在于使用了先进的电化学检测方法来量化临床样本中逆转录酶(RT)的活性。病毒逆转录酶在病毒复制中起着至关重要的作用，与HIV亚型无关。由于该酶的存在与病毒RNA的存在高度相关，RT活性分析是一种公认的(但未经FDA批准)监测病毒载量的替代方法。与难以量化某些HIV亚型经常变化的RNA的基于PCR的检测不同，RT活性检测对HIV-1(无论亚型)和HIV-2同样有效，可以在没有高技能技术人员的情况下完成，而且不太容易受到污染问题的影响。Alderon的第一阶段工作表明，电化学方法量化病毒RT活性是可行的，与光学分析相比，有可能极大地提高性能(每次测试的时间和成本)。Alderon提出的为期3年的第二阶段SBIR项目有4个具体目标，这些目标将导致电子系统的改进，并在方便和低成本的当天测试中准确检测艾滋病毒载量。目标1和目标2的重点是改进仪器和传感器，使病毒载量分析更负担得起，在资源有限的情况下使用起来更简单。目的3试剂和检测方法的改进将进一步减少检测时间和复杂性。目标4在广泛样本的病毒负载测试中验证eSystem的性能将为FDA批准和产品商业化奠定基础。与北卡罗来纳大学艾滋病研究中心的合作将建立eSystem检测结果与通过FDA批准的替代方法获得的病毒载量结果之间的相关性。将通过与麦哲伦生物科学公司的合作促进产品商业化，麦哲伦生物科学公司的工程专业知识将帮助开发可靠和坚固的仪器和病毒负载测试试剂盒组件，这些组件可以在经过良好制造做法认证的设施中生产。公共卫生相关性：据估计，全世界目前有3320万艾滋病毒携带者，2007年它夺走了210万人的生命，其中包括33万儿童(世界卫生组织)。为了满足对艾滋病毒感染者更好的治疗和诊断检测的迫切需求，Alderon Biosciences，Inc.建议进一步开发一种低成本和易于使用的电化学测试系统(ESystem)，用于确定临床样本中的艾滋病毒病毒载量。Alderon的方法将使在初级保健或护理点(POC)环境中进行病毒负载测试成为可能，并有助于在资源有限的环境中监测和治疗艾滋病毒阳性患者。

DESCRIPTION (provided by applicant): To address the urgent need for better treatment and diagnostic testing of HIV infected people in resource-limited settings, we propose in this Phase II SBIR to further develop an electrochemical testing system (eSystem) developed by Alderon Biosciences, Inc. for determinations of HIV viral load in clinical samples. Alderon's long-term goal is to produce, validate, and gain FDA approval for a facile and affordable (< $10) HIV viral load test, sensitive to below 500 copies/ml, that gives same-day results using an inexpensive and portable instrument. Currently available FDA-approved HIV viral load tests require expensive instrumentation, utilize complex molecular biology procedures, and can cost > $100 per test. Our hypothesis is that Alderon can develop a far more facile and affordable system for accurate viral load measurements and drug susceptibility testing that will enable viral load testing to be done in resource-limited point-of-care (POC) environments. The innovation in the new HIV viral load tests proposed by Alderon lies in the use of advanced electrochemical detection methods for quantifying the activity of reverse transcriptase (RT) in clinical samples. The viral RT enzyme plays an essential role in viral replication, regardless of HIV subtype. Because the enzyme's presence is highly correlated with that of the viral RNA, RT activity assays are a recognized (but not FDA-approved) alternative method for monitoring viral load. Unlike PCR-based assays that have problems quantifying the often-changing RNAs of some HIV subtypes, RT activity tests work equally well for HIV-1 (regardless of subtype) and HIV-2, can be done without highly skilled technicians, and are less subject to problems of contamination. Alderon's Phase I work has shown feasibility of an electrochemical approach to quantification of viral RT activity, with the potential for greatly improved performance (time and cost per test) relative to optical assays. Alderon's proposed 3 year Phase II SBIR project has 4 Specific Aims that will lead to eSystem improvements and accurate detection of HIV load in facile and low-cost same-day tests. Aims 1 and 2 focus on improvements in the instrument and sensors that will make the viral load assays more affordable and simpler for use in resource-limited settings. Aim 3 improvements of reagents and assay protocols will further reduce assay time and complexity. Aim 4 validation of eSystem performance in viral load tests with a wide range of samples will set the stage for FDA approval and product commercialization. Collaborative work with the UNC Center for AIDS Research will establish the correlation between results of eSystem assays and viral load results obtained by alternative FDA- approved methods. Product commercialization will be facilitated by collaborative work with Magellan Biosciences, whose engineering expertise will aid in the development of reliable and robust instruments and viral load test kit components that can be produced in facilities certified for good manufacturing practices. PUBLIC HEALTH RELEVANCE: An estimated 33.2 million people are now living with HIV disease worldwide and in 2007 it claimed the lives 2.1 million people, including 330,000 children (World Health Organization). To address the urgent need for better treatment and diagnostic testing of HIV infected people, Alderon Biosciences, Inc. proposes to further develop a low-cost and easy-to-use electrochemical testing system (eSystem) for determinations of HIV viral load in clinical samples. Alderon's approach will make it possible to perform viral load testing in primary care or point-of-care (POC) environments and aid in monitoring and treating HIV positive patients in resource-limited environments.