Development of CNS-targeted AAV vectors

CNS靶向AAV载体的开发

• 批准号: 8130697
• 负责人: MIGUEL S ESTEVES
• 金额: $ 32.24万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8130697
• 关键词: Achievement; Address; Adult; Affect; Amino Acids; Animals; Antibodies; Area; Award; Back; Biochemical; Biodistribution; Bioluminescence; Biotin; Brain; Capsid; Cerebrum; Childhood; Clinical; Clinical Trials; Communities; DNA; DNA Shuffling; Development; Elderly; Electronics; Environment; Enzymes; Firefly Luciferases; Fostering; Funding; Gangliosidosis GM1; Gene Delivery; Gene Expression; Generations; Genes; Grant; Guidelines; Health; Human; Human Resources; Image; Infusion procedures; Intravenous; Intravenous infusion procedures; Kinetics; Leadership; Libraries; Liposomes; Logic; Lysosomal Storage Diseases; Mediating; Methodology; Methods; Modern Medicine; Molecular; Molecular Bank; Mus; National Institute of Drug Abuse; National Institute of Mental Health; National Institute of Neurological Disorders and Stroke; Organ; Outcome; Peptides; Persons; Phase; Plasmids; Population; Probability; Process; Program Reviews; Property; Proteins; Published Comment; Recombinants; Recording of previous events; Research; Research Personnel; Role; Spinal Cord; Streptavidin; Tail; Technology; Testing; TimeLine; Tissues; Training; Transferrin Receptor; Treatment Efficacy; United States National Institutes of Health; Veins; Vertebral column; Virion; Work; abstracting; adeno-associated viral vector; apolipoprotein B-100; base; cellular transduction; clinical practice; design; effective therapy; gene therapy; in vivo; innovation; lateral ventricle; mature animal; meetings; mouse model; nervous system disorder; novel; organizational structure; rabies virus glycoprotein G; relating to nervous system; success; tool; transgene expression

DESCRIPTION (provided by applicant): Abstract - AAV vectors are exceptionally efficient for gene delivery to the brain by direct intraparenchymal infusion, where they mediate continuous transgene expression perhaps for the lifetime of the experimental animal. These exceptional properties of existing AAV vectors are the basis for ongoing or planned clinical trials for neurological diseases where focal gene delivery is therapeutically effective. However development of gene therapy approaches for many other neurological diseases will require global gene delivery to the CNS. Here we will use in vivo selection of an AAV capsid library and molecular grafting to develop new CNS- targeted AAV vectors with such capability. In the first approach we will use in vivo selection of an AAV capsid library with a diversity of 5x109 clones generated by DNA shuffling of AAV1, 2, 5, 8, 9, and rh10 Cap genes, to identify new brain- and spinal cord-tropic AAV capsids after intravenous or ICV delivery in adult animals. The library will be infused via the tail vein or the cerebral lateral ventricles of adult mice and one month later we will isolate DNA from the brain and spinal cord for PCR amplification of tissue-resident AAV Cap genes. These will be cloned back into the original library plasmid backbone and used to produce more AAV virions for subsequent rounds of in vivo selection. We will sequence 10 AAV Cap genes per round of selection. Once we determine convergence of tissue resident AAV Cap genes for each target, we will prepare recombinant AAV vectors encoding firefly luciferase (Fluc) using the newly selected capsids and infuse them into adult animals to determine their biodistribution. We will use bioluminescence imaging to assess distribution and kinetics of gene expression followed by biochemical quantification of Fluc activity in different organs and histological assessment of transduced cell distribution in brain and spinal cord. In the second approach we will combine AAV vectors with different proteins and peptides previously shown to be highly efficient in ferrying liposomes, enzymes, and siRNAs across the BBB after intravascular infusion. For this we will use chimeric AAV capsids carrying a 14 amino acid biotin acceptor peptide that allows for incorporation of biotin into the AAV capsid during packaging. As brain-targeting molecules we will use Streptavidin fused to a single-chain antibody specific for the mouse Transferrin receptor (TfR), or peptides derived from human ApoB-100, or Rabies virus glycoprotein. The biodistribution of the new AAV-molecular conjugates after intravenous infusion will be assessed as above. The therapeutic efficacy of all new CNS-targeted AAV vectors will be tested in a mouse model of GM1-gangliosidosis, which is a lysosomal storage disease that severely affects the CNS. We expect this new generation of CNS-targeted AAV vectors to foster the development of highly effective gene therapy approaches to treat many childhood, adult, and geriatric neurological diseases currently beyond the reach of modern medicine PUBLIC HEALTH RELEVANCE: The purpose of this proposal is to develop a new generation of AAV vectors capable of targeting the brain via the vasculature. Achieving global gene delivery to the adult CNS remains the 'holy grail' of neural gene therapy, and it is likely necessary to develop effective therapies for many neurological diseases. Here we will investigate two approaches to develop new CNS-targeted AAV vectors: 1) In vivo selection of an AAV capsid library via intravascular and intracerebroventricular delivery in adult animals; 2) AAV-molecular conjugates incorporating brain-tropic proteins and peptides. This new generation of AAV vectors may provide the means to develop highly effective gene therapy approaches to treat many childhood, adult, and geriatric neurological diseases currently beyond the reach of modern medicine. The NIDA/NIMH/NINDS EUREKA applications were reviewed differently from more traditional NIH grant mechanisms. Specifically, the review process consisted of two phases. During the first (i.e., electronic) phase a selected panel of reviewers were given the following guidelines by which to assess the applications. They were asked to determine whether they: Strongly Agree, Moderately Agree, Neither Agree nor Disagree, Moderately Disagree, or Strongly Disagree with these descriptions. Their ratings and any additional comments are below. These initial ratings also provided the basis for the review panel to determine whether an application would be discussed during an in person meeting. Because of the very stringent review criteria and limited pool of funds set aside for this program, the review panel chose only to discuss applications that garnered the most enthusiasm. The Resume and Summary of the Discussion above summarizes opinions of the person meeting and forms the basis of the final score. Significance: This study addresses an important problem and the outcome of the proposed studies will drive the field. The potential impact of the proposed research is exceptional, in terms of the magnitude of the impact and the size of the community affected. Innovation: The project is highly original and exceptionally innovative and seriously challenges existing paradigms or clinical practice. The project addresses a major barrier to progress in the field or it develops or employs exceptionally novel concepts, approaches, methodologies, tools, or technologies. Approach: The logic of the approach is sufficiently compelling despite the lack of experimental detail. The conceptual (or clinical) framework, design, methods, and analyses are adequately developed, well integrated and reasoned, and are appropriate for the aims of the project. The applicant acknowledges potential problem areas and considers alternative tactics. The information in the timeline inspires confidence that the PI will be able to document progress in each year of the award and either complete the project or demonstrate conclusively that it cannot be completed, despite good-faith efforts, during the term of the award. The requested duration of the award is appropriate for the proposed research. Investigators: The PD/PI(s) and other key personnel are appropriately trained and well-suited to carry out this work. Past achievements of the PI(s) suggest that the investigator(s) is/are exceptionally innovative and likely to make paradigm-shifting, high-impact discoveries. If the PI does not have a history of doing exceptionally innovative, high-impact research, the logic of the experimental plan suggests that there is at least some likelihood of success. The project is high priority for the PI(s), as indicated by the person-months of effort that the PI(s) will devote to it. For applications designating multiple PDs/PIs, the leadership plan, including the designated roles and responsibilities, governance, and organizational structure, are consistent with and justified by the aims of the project and the expertise of each of the PDs/PIs. Environment: The scientific environment(s), in which the work will be performed, contributes to the probability of success. The proposed studies benefit from unique features of the scientific environment, subject populations, or employ useful collaborative arrangements. There is evidence of institutional support.

描述（由申请人提供）： 摘要 - AAV载体通过直接的核内输注对基因递送至大脑具有极有效的效率，在那里它们介导了连续的转基因表达，这可能是实验动物的寿命。现有AAV载体的这些特殊特性是对焦点基因递送在治疗上有效的神经系统疾病的临床试验或计划的临床试验的基础。但是，许多其他神经系统疾病的基因治疗方法的发展将需要向中枢神经系统递送全球基因。在这里，我们将在体内选择AAV CAPSID库和分子移植，以开发具有这种能力的新的CNS靶向AAV矢量。在第一种方法中，我们将使用体内选择AAV CapSID库，其多样性为5x109克隆，该克隆通过AAV1、2、5、5、8、9和RH10 CAP基因的DNA改组来识别成人动物的静脉或ICV递送后，识别新的脑和脊髓 - 脊髓 - 热带AAV CAPSID。该文库将通过尾静脉或成年小鼠的脑外侧心室注入，一个月后，我们将从脑和脊髓中分离DNA，以扩增组织居住的AAV CAP基因。这些将被克隆回原始的库质粒主链，并用于生产更多的AAV病毒体，用于随后的体内选择。我们将每一轮选择序列10 AAV CAP基因。一旦我们确定了每个目标的组织驻留AAV CAP基因的收敛性，我们将使用新选择的衣壳编码编码萤火虫荧光素酶（FLUC）的重组AAV载体，并将其注入成年动物中，以确定其生物分布。我们将使用生物发光成像评估基因表达的分布和动力学，然后对不同器官中FLUC活性的生化定量以及对脑和脊髓中细胞分布的组织学评估。在第二种方法中，我们将将AAV载体与先前证明的不同蛋白质和肽相结合，在血管内输注后横跨BBB的脂质体，酶和siRNA高效。为此，我们将使用携带14个氨基酸生物素受体肽的嵌合AAV衣壳，在包装过程中允许将生物素掺入AAV CAPSID中。作为脑靶向分子，我们将使用链霉亲和素融合到针对小鼠转铁蛋白受体（TFR）的单链抗体或源自人APOB-100或狂犬病病毒糖蛋白的肽。静脉输注后新的AAV分子共轭物的生物分布将如上评估。所有新型CNS靶向AAV载体的治疗功效将在GM1 - 旋转病的小鼠模型中进行测试，GM1 - 长方体病的小鼠模型是一种严重影响CNS的溶酶体储存疾病。我们期望新一代的CNS靶向AAV媒介来促进高效的基因疗法方法来治疗许多童年，成人和老年神经系统疾病，目前目前超出了现代医学的影响 公共卫生相关性： 该提案的目的是开发新一代的AAV向量，能够通过脉管系统靶向大脑。实现向成年中枢神经系统的全球基因递送仍然是神经基因疗法的“圣杯”，可能有必要为许多神经疾病开发有效的疗法。在这里，我们将研究两种开发新的CNS靶向AAV载体的方法：1）通过血管内和脑内脑室递送在成年动物中，体内选择AAV衣壳文库； 2）AAV分子缀合物结合了脑循环蛋白和肽。这一新一代的AAV媒介可能会提供开发高效的基因疗法方法来治疗目前超出现代医学范围的许多童年，成人和老年神经系统疾病。 NIDA/NIMH/NINDS EUREKA应用程序的审查与更传统的不同 NIH赠款机制。具体而言，审查过程由两个阶段组成。在 首先，（即电子）阶段A选定的审阅者面板由以下指南。 评估申请。他们被要求确定他们是否：完全同意， 中度同意，既不同意也不同意，中度不同意或强烈不同意 这些描述。他们的评分和任何其他评论如下。这些初始评分 还为审核面板提供了确定申请是否将是的基础 在一次会议上讨论。由于非常严格的评论标准和有限 为该计划拨出的资金库，评论面板只选择讨论申请 这赢得了最热情。上面讨论的简历和摘要 总结人会议的意见，并构成最终得分的基础。 意义：本研究解决了一个重要的问题，拟议研究的结果将 驱动场。就幅度而言，拟议研究的潜在影响是非凡的 社区影响的影响和大小受到影响。 创新：该项目是高度原始和异常创新和严重挑战的现有 范式或临床实践。该项目解决了该领域进步或IT的主要障碍 开发或采用异常新颖的概念，方法，方法，工具或技术。 方法：尽管缺乏实验细节，但该方法的逻辑足以令人信服。 概念（或临床）框架，设计，方法和分析已充分发展，很好 集成和推理，适合项目的目的。申请人承认 潜在的问题领域并考虑替代策略。时间表中的信息灵感 对PI的信心将能够记录奖励的每一年的进度并完成 该项目或最终证明，尽管有良好的努力，但在 奖项的期限。该奖项的持续时间适合拟议的研究。 调查人员：PD/PI（S）和其他关键人员经过适当培训，适合携带 出去这项工作。 PI的过去成就表明，研究者是/异常的 创新的，可能会带来范式转移，高影响力的发现。如果PI没有 进行异常创新，高影响研究的历史，实验计划的逻辑 表明至少有成功的可能性。该项目是PI的优先事项 PI将致力于这一努力的努力表明。用于指定的应用 多个PDS/PI，领导计划，包括指定的角色和职责，治理， 和组织结构与项目的目的一致和合理 每个PDS/PI的专业知识。 环境：将执行工作的科学环境，有助于 成功的概率。拟议的研究受益于科学环境的独特特征， 主题人群，或采用有用的协作安排。有制度的证据 支持。