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Rational and Combinatorial Engineering of AAV Vectors

AAV载体的合理和组合工程

基本信息

批准号：
8660593
负责人：
Matthew Louis Hirsch
金额：
$ 37.81万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-12-01 至 2018-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8660593
关键词：
Address Alternative Splicing Animal Model Antisense Oligonucleotides Atlases Binding Biology Capsid Cell Surface Receptors Cell model Clinic Clinical Clinical Data Clinical Trials DNA DNA Repair DNA Repair Pathway DNA delivery Data Databases Disease Dose Duchenne muscular dystrophy Engineering Event Exhibits Eye Factor IX Gene Delivery Gene Expression Regulation Gene Transduction Agent Genetic Recombination Genome Heart Hemophilia A Hereditary Disease Human Human Cell Line Immune In Vitro Introns Investigation Liver Maps Mediating Methods Molecular Mouse Cell Line Mus Nature Nonhomologous DNA End Joining Organ Outcome Performance Prevalence Primates Process Production Proteins Publications Reagent Reliance Reporting Serotyping Single-Stranded DNA Structure-Activity Relationship System Systemic disease Technology Tissue Sample Tissues Titrations Transgenes Transgenic Organisms Triage Variant Viral Work adeno-associated viral vector base clinical efficacy combinatorial density design directed evolution disease phenotype gene therapy genetic manipulation homologous recombination human disease human tissue in vivo insight mouse model mutant nonhuman primate novel pre-clinical protein expression public health relevance receptor receptor binding receptor expression repaired research study therapeutic protein tissue tropism transgene expression vector

项目摘要

DESCRIPTION (provided by applicant): The available data of AAV vectors in the clinic emphasize the importance of continued optimization efforts at the levels of the AAV capsid, genome and transgenic cassette. A focus of this proposal is to derive clinical AAV vector best suited for systemic disorders (MPS, Hurlers, etc.). At the capsid level, it is apparent that animal models do not always predict the human outcome and that more efficient human specific capsids are required to achieve a lower administered dose. In Aim 1, we seek to create a new paradigm of AAV vector selection for human transduction by generating the first AAV receptor expression map on tissues of mouse, primate and human origin. This tissue specific AAV receptor Atlas will be overlaid with AAV binding and transduction data in an effort to tease out regions of the capsid important for tissue specific interactions in varied backgrounds. In addition novel chimeric capsids isolated from a directed evolution strategy on primate and human livers established in a mouse model will be triaged against our receptor/binding atlas to determine if in vitro binding correlates to in vivo results. Then, capsid isolates from a primatized-liver mouse model will be investigated for primate liver transduction in vivo to determine if this strategy represents a valid method to derive primate (human & non human) liver specific AAV capsids. At the level of the AAV genome, we have assembled a panel of DNA repair dependent AAV substrates that report critical aspects of genome persistence including circularization, concatemerization and homology directed annealing. Investigations of these reagents in mutant backgrounds defective in different DNA repair pathways will offer insights into the preferred reliance on homologous recombination and non-homologous end joining mechanisms in vitro and in vivo providing a better prediction of vector performance in diseased settings (Aim 2). At the level of the vector transgene, we demonstrate in mouse liver, heart and eye a novel method to induce transgene synthesis using the IVS2- 654 intron and an anti-sense oligonucleotide. The work herein seeks to generate smaller synthetic variants that exhibit tighter control as well as altered transgene expression levels, thus providing a panel of regulatory switches which can be tailored for specific applications. Finally, a strategy is proposed to engineer an "off" switch for the induced transgene synthesis from IVS2-654, which may also allow the precise tuning of transgene synthesis at a fixed vector dose. Collectively, the results of the proposed experiments seek to address the observed clinical deficiencies in AAV gene therapy applications for diseases of systemic nature by our continued optimization efforts at the levels of the capsid and genome as well as the transgenic DNA cassette.

描述（由申请人提供）：AAV载体在临床中的可用数据强调了在AAV衣壳、基因组和转基因盒水平上持续优化工作的重要性。该提案的重点是衍生最适合全身性疾病（MPS、Hurlers 等）的临床 AAV 载体。在衣壳水平上，很明显动物模型并不总能预测人类结果，并且需要更有效的人类特异性衣壳才能实现较低的给药剂量。在目标 1 中，我们寻求通过在小鼠、灵长类动物和人类起源的组织上生成第一个 AAV 受体表达图来创建用于人类转导的 AAV 载体选择的新范例。该组织特异性 AAV 受体图谱将覆盖 AAV 结合和转导数据，以努力梳理出对不同背景下的组织特异性相互作用重要的衣壳区域。此外，从小鼠模型中建立的灵长类动物和人类肝脏的定向进化策略中分离出的新型嵌合衣壳将根据我们的受体/结合图谱进行分类，以确定体外结合是否与体内结果相关。然后，将从灵长类动物肝脏小鼠模型中分离的衣壳进行体内灵长类动物肝脏转导的研究，以确定该策略是否代表衍生灵长类动物（人类和非人类）肝脏特异性 AAV 衣壳的有效方法。在 AAV 基因组水平上，我们组装了一组 DNA 修复依赖性 AAV 底物，报告基因组持久性的关键方面，包括环化、串联和同源定向退火。在不同 DNA 修复途径缺陷的突变体背景中对这些试剂的研究将深入了解体外和体内同源重组和非同源末端连接机制的首选依赖，从而更好地预测患病环境中的载体性能（目标 2）。在载体转基因水平上，我们在小鼠肝脏、心脏和眼睛中展示了一种使用 IVS2-654 内含子和反义寡核苷酸诱导转基因合成的新方法。本文的工作旨在产生更小的合成变体，这些变体表现出更严格的控制以及改变的转基因表达水平，从而提供一组可以针对特定应用定制的调节开关。最后，提出了一种策略来设计一个“关闭”开关 IVS2-654 诱导的转基因合成，这也可能允许在固定载体剂量下精确调整转基因合成。总的来说，所提出的实验结果旨在通过我们在衣壳和基因组以及转基因 DNA 盒水平上的持续优化努力，解决系统性疾病的 AAV 基因治疗应用中观察到的临床缺陷。