权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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：
8578289
负责人：
Matthew Louis Hirsch
金额：
$ 35.54万
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/8578289
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）：临床中AAV载体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。该提议的重点是获得最适合于全身性疾病（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是预测人的结果，并且需要更有效的人特异性衣壳来实现较低的施用剂量。在目标1中，我们试图通过在小鼠、灵长类动物和人来源的组织上产生第一个AAV受体表达图谱来创建用于人转导的AAV载体选择的新范例。该组织特异性AAV受体图谱将与AAV结合和转导数据重叠，以努力梳理出对不同背景中的组织特异性相互作用重要的衣壳区域。此外，从在小鼠模型中建立的灵长类动物和人肝脏的定向进化策略中分离的新型嵌合衣壳将针对我们的受体/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，将研究来自灵长类动物化肝脏小鼠模型的衣壳分离物的体内灵长类动物肝脏转导，以确定该策略是否代表衍生灵长类动物（人和非人）肝脏特异性AAV衣壳的有效方法。在AAV基因组水平，我们已经组装了一组DNA修复依赖性AAV底物，其报告了基因组持久性的关键方面，包括环化、多联体化和同源性定向退火。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提供对体外和体内同源重组和非同源末端连接机制的优选依赖性的见解，从而提供对疾病环境中载体性能的更好预测（目的2）。在载体转基因的水平上，我们在小鼠肝脏、心脏和眼睛中展示了一种使用IVS 2 - 654内含子和反义寡核苷酸诱导转基因合成的新方法。本文的工作试图产生较小的合成变体，其表现出更严格的控制以及改变的转基因表达水平，从而提供一组可针对特定应用定制的调控开关。最后，提出了一种策略，以设计一个“关闭”开关，从IVS 2 -654诱导转基因合成，这也可以允许在固定载体剂量下精确调节转基因合成。总的来说，所提出的实验的结果试图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来解决在用于全身性疾病的AAV基因治疗应用中观察到的临床缺陷。