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 载体通过直接脑实质内输注将基因输送到大脑非常有效，它们可能在实验动物的一生中介导持续的转基因表达。现有 AAV 载体的这些特殊特性是正在进行或计划中的神经系统疾病临床试验的基础，其中局部基因递送具有治疗效果。然而，许多其他神经系统疾病的基因治疗方法的开发将需要将基因递送至中枢神经系统。在这里，我们将利用 AAV 衣壳库的体内选择和分子移植来开发具有这种能力的新的 CNS 靶向 AAV 载体。在第一种方法中，我们将使用 AAV 衣壳文库的体内选择，该文库具有通过 AAV1、2、5、8、9 和 rh10 Cap 基因的 DNA 改组生成的多样性 5x109 克隆，以在成年动物静脉内或 ICV 递送后识别新的脑和脊髓亲和性 AAV 衣壳。该文库将通过成年小鼠的尾静脉或大脑侧脑室注入，一个月后我们将从大脑和脊髓中分离 DNA，用于组织驻留 AAV Cap 基因的 PCR 扩增。这些将被克隆回原始文库质粒主链，并用于产生更多 AAV 病毒颗粒，用于后续几轮体内选择。我们将对每轮选择的 10 个 AAV Cap 基因进行测序。一旦我们确定了每个靶标的组织驻留 AAV Cap 基因的收敛性，我们将使用新选择的衣壳制备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体内以确定其生物分布。我们将使用生物发光成像来评估基因表达的分布和动力学，然后对不同器官中的 Fluc 活性进行生化定量，并对大脑和脊髓中转导细胞的分布进行组织学评估。在第二种方法中，我们将 AAV 载体与不同的蛋白质和肽结合起来，这些蛋白质和肽先前已被证明在血管内输注后能够高效地跨 BBB 运送脂质体、酶和 siRNA。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，该肽允许在包装过程中将生物素掺入 AAV 衣壳中。作为脑靶向分子，我们将使用与小鼠转铁蛋白受体 (TfR) 特异性单链抗体融合的链霉亲和素，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-分子缀合物的生物分布将如上所述进行评估。所有新的针对中枢神经系统的 AAV 载体的治疗效果将在 GM1 神经节苷脂沉积症小鼠模型中进行测试，这是一种严重影响中枢神经系统的溶酶体贮积病。我们期望新一代靶向中枢神经系统的 AAV 载体能够促进高效基因治疗方法的开发，以治疗目前现代医学无法达到的许多儿童、成人和老年神经系统疾病 公共卫生相关性： 该提案的目的是开发能够通过脉管系统靶向大脑的新一代 AAV 载体。实现成人中枢神经系统的整体基因传递仍然是神经基因治疗的“圣杯”，并且可能有必要开发针对许多神经系统疾病的有效疗法。在这里，我们将研究两种开发新的 CNS 靶向 AAV 载体的方法：1）通过成年动物血管内和脑室内递送体内选择 AAV 衣壳库； 2) 包含亲脑蛋白和肽的 AAV 分子缀合物。新一代 AAV 载体可能提供开发高效基因治疗方法的手段，以治疗目前现代医学无法达到的许多儿童、成人和老年神经系统疾病。 NIDA/NIMH/NINDS EUREKA 申请的审核方式与传统申请不同 NIH 资助机制。具体来说，审查过程分为两个阶段。期间 第一（即电子）阶段，选定的评审小组获得了以下指导方针： 用于评估应用程序。他们被要求确定是否： 强烈同意， 比较同意、既不同意也不反对、比较不同意或强烈不同意 这些描述。他们的评级和任何其他评论如下。这些初始评级 还为审查小组确定申请是否得到批准提供了依据 在面对面会议期间进行讨论。由于审查标准非常严格且数量有限 为该计划预留的资金池，审查小组选择仅讨论申请 这引起了最大的热情。上述讨论的简历和总结 总结会议成员的意见，形成最终评分的基础。 意义：这项研究解决了一个重要问题，拟议研究的结果将 驱动领域。就规模而言，拟议研究的潜在影响是非凡的 影响以及受影响社区的规模。 创新：该项目具有高度原创性和非凡的创新性，严重挑战现有的 范式或临床实践。该项目解决了该领域取得进展的主要障碍 开发或采用异常新颖的概念、途径、方法、工具或技术。 方法：尽管缺乏实验细节，但该方法的逻辑足够引人注目。 概念（或临床）框架、设计、方法和分析得到充分发展、良好 综合且合理，并且适合项目的目标。申请人承认 潜在的问题领域并考虑替代策略。时间轴上的信息鼓舞人心 相信 PI 将能够记录获奖每一年的进展，并且完成 该项目或最终证明，尽管有诚意的努力，该项目仍无法完成 奖励期限。所要求的奖励期限适合拟议的研究。 调查人员：PD/PI 和其他关键人员经过适当的培训并且非常适合携带 出这个作品。 PI 过去的成就表明研究者非常出色 创新并可能做出范式转变、高影响力的发现。如果 PI 没有 进行异常创新、高影响力研究的历史、实验计划的逻辑 表明至少有一定的成功可能性。该项目对于 PI 来说具有高度优先级，因为 由PI（们）为此付出的人月努力来表示。对于指定的应用 多个 PD/PI、领导力计划，包括指定的角色和职责、治理、 和组织结构，与项目和项目的目标一致并合理 每个 PD/PI 的专业知识。 环境：开展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， 受试者群体，或采用有用的合作安排。有证据表明制度存在 支持。