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：
9060877
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/9060877
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 cassette DNA delivery Data Databases Directed Molecular Evolution Disease Dose Duchenne muscular dystrophy Engineering Event Exhibits Eye Factor IX Gene Delivery Gene Expression Regulation Gene Transduction Agent Genetic Recombination Genome Health 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 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 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 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（申请人提供）：诊所中AAV矢量的可用数据强调了在AAV CAPSID，基因组和转基因盒的水平上持续优化工作的重要性。该提案的重点是得出最适合全身性疾病（国会议员，投掷者等）的临床AAV载体。在衣壳级上，很明显动物模型并不总是预测人类的结果，并且需要更有效的人类特异性衣壳才能达到较低的剂量。在AIM 1中，我们试图通过在小鼠，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AAV矢量选择的新范式。该组织特异性AAV受体地图集将用AAV结合和转导数据覆盖，以促进在各种背景下对组织特异性相互作用重要的衣壳区域。此外，从针对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出来的新型嵌合胶囊将与我们的受体/结合图集进行分解，以确定体外结合是否与体内结果相关。然后，将研究从肝小鼠模型中的衣壳分离株进行体内灵长类动物肝转导的转导，以确定该策略是否代表了推导灵长类动物（人类和非人类）肝脏特异性AAV Capsids的有效方法。在AAV基因组的水平上，我们组装了一组DNA修复依赖的AAV底物，这些依赖性的AAV底物报告了基因组持久性的关键方面，包括循环，屈服和指示退火的同源性。在不同DNA修复途径中有缺陷的突变背景中这些试剂的研究将为您提供对同源重组和非同源末端连接机制的首选依赖的见解，并在体内提供了更好地预测患病环境中矢量性能的方法（AIM 2）。在矢量转基因的水平上，我们在小鼠肝脏，心脏和眼睛中证明了一种新的方法，是使用IVS2- 654内含子和抗义寡核苷酸诱导转基因合成的新方法。这里的工作旨在生成较小的合成变体，这些变体表现出更严格的控制以及改变转基因表达水平，从而提供了一系列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”开关从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，提出的实验的结果试图解决AAV基因治疗应用中观察到的全身性质疾病中观察到的临床缺陷，这是通过我们在衣壳和基因组水平以及转基因DNA录音水平上的持续优化工作。