Next Generation of Recombinant AAV Serotype Vectors for Gene Therapy

用于基因治疗的下一代重组 AAV 血清型载体

• 批准号: 8251153
• 负责人: Roland W. Herzog
• 金额: $ 61.37万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-15 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8251153
• 关键词: Adenovirus Vector; Adenoviruses; Animal Model; Applications Grants; Attention; Baculoviruses; Biology; Canis familiaris; Capsid; Capsid Proteins; Cell Communication; Cell Nucleus; Cells; DNA; Dependovirus; Development; Disease; Dose; Evaluation; Exhibits; Factor IX; Gene Transduction Agent; Genes; Helper Viruses; Hemophilia A; Hemophilia B; Hemorrhage; Hepatocyte; Herpesviridae; Human; Immune response; Immunology; In Vitro; Knockout Mice; Knowledge; Lead; Life Cycle Stages; Light; Liver; Liver diseases; Mediating; Modeling; Molecular; Mus; Muscle; Mutation; Nature; Parkinson Disease; Parvovirus; Patients; Phase; Production; Property; Public Health; Recombinant adeno-associated virus (rAAV); Retroviral Vector; Retroviridae; Safety; Serotyping; Single-Stranded DNA; Site; System; Techniques; Testing; Therapeutic; Tissues; Transgenes; Transgenic Mice; Trypsin; Tyrosine; Vaccinia virus; Viral Genome; Virus; abstracting; adeno-associated viral vector; base; cell type; clinical efficacy; comparative; gene therapy; gene therapy clinical trial; in vivo; insight; interest; latent infection; multicatalytic endopeptidase complex; mutant; next generation; novel; public health relevance; stable cell line; therapeutic gene; trafficking; transduction efficiency; transgene expression; tumor; vector

DESCRIPTION (provided by applicant): Project Summary/Abstract The main aims of this multiple-PI proposal are to evaluate the safety and eficacy of, and the host immune response to, the next generation of recombinant adeno-associated virus (AAV) vectors that we have developed, in small and large animal models of human liver diseases in general, and hemophilia in particular. AAV vectors have gained attention as an alternative to the more commonly used retrovirus and adenovirus vectors, and are in use in Phase I/II clinical trials for gene therapy of a number of diseases. However, relatively large vector doses are needed to achieve therapeutic benefits. Large vector doses also trigger an immune response as a significant fraction of the vectors fails to traffic efficiently to the nucleus, and is targeted for degradation by the host cell proteasome machinery. Our recent studies have yielded insights into key steps in intracellular trafficking of AAV, and led to the development of novel AAV vectors that are capable of high-efficiency transduction at lower doses. We will test the following hypotheses: a. Combination of specific tyrosine mutations in AAV2 capsids will further reduce the vector dose needed for high-efficiency transduction, and corresponding mutations in tyrosine residues in AAV8 and AAV5 serotype vectors will lead high-efficiency transduction of murine and canine hepatocytes. b. Novel Baculovirus system-produced rAAV vectors, characterized by higher VP1 capsid protein stoichiometric content, will exhibit superior transduction properties in target tissues. c. Tyrosine-mutant AAV vectors will elicit a reduced host cell immune response, and provide therapeutic benefits at lower doses. The following three Specific Aims will be pursued: 1. Development of AAV2 vectors containing multiple tyrosine-mutations, elucidation of the underlying mechanism of transduction by the most efficient vector in vitro and in vivo, and comparative analysis with AAV8 and AAV5 vectors. 2. Development of the next generation of Sf9-based stable cell lines for the production of highly infectious rAAV of alternative serotypes. 3. Treatment of murine and canine hemophilia B with optimal tyrosine-mutant AAV2, AAV8, and AAV5 serotype vectors and evaluation of immune responses to vector and coagulation factor IX transgene product. The knowledge gained from these studies will not only shed light on the AAV-host cell interactions, but will also be applicable in further improvements in recombinant AAV vectors for their potential use in gene therapy of human liver diseases in general, and hemophilia in particular. PUBLIC HEALTH RELEVANCE: Project Narrative The main aim of this proposal is to develop the next generation of vectors with which a therapeutic gene can be safely delivered to patients with a bleeding disorder called hemophilia B. These vectors are derived from a virus that causes no known disease, and is therefore, expected to be safer. The development of such a vector for the potential treatment and cure of hemophilia therefore has relevance to public health.

描述（由申请人提供）： 项目摘要/摘要这一多PI提案的主要目的是评估我们在一般人类肝脏疾病的小型和大型动物模型中开发的下一代重组腺相关病毒（AAV）向量的安全性和宿主免疫反应的安全性和效率。 AAV载体已引起人们的注意，以替代更常用的逆转录病毒和腺病毒载体，并正在使用用于多种疾病的基因治疗的I/II期临床试验中。但是，需要相对较大的矢量剂量来获得治疗益处。大型矢量剂量还会触发免疫反应，因为矢量的很大一部分无法有效地传输到核，并且是由宿主细胞蛋白酶体机械降解的。我们最近的研究已经深入了解了AAV细胞内贩运的关键步骤，并导致了新型AAV载体的发展，而AAV载体能够在较低剂量下进行高效转导。我们将测试以下假设： AAV2衣壳中特定酪氨酸突变的组合将进一步减少高效转导所需的载体剂量，以及AAV8和AAV5血清型矢量中酪氨酸残基中相应的突变将导致鼠和犬肝细胞的高效转导。 b。新型的杆状病毒系统产生的RAAV载体，其特征在于较高的VP1衣壳蛋白化学计量含量，将在靶组织中表现出较高的转导特性。 c。酪氨酸突出的AAV载体将引起降低的宿主细胞免疫反应，并以较低剂量提供治疗益处。 将实现以下三个特定目标：1。含有多种酪氨酸突变的AAV2载体的发展，最有效的载体在体外和体内阐明了转导的基本机制，以及对AAV8和AAV5载体的比较分析。 2。基于SF9的稳定细胞系的开发，用于产生高度感染性的替代血清型。 3。用最佳的酪氨酸突变剂AAV2，AAV8和AAV5血清型矢量处理鼠和犬血友病B，以及对载体和凝结因子IX转基因产物的免疫反应的评估。 从这些研究中获得的知识不仅会阐明AAV宿主 - 宿主相互作用，而且还将适用于重组AAV媒介的进一步改善，以便在通常的基因治疗人肝病的基因治疗中，特别是血友病。 公共卫生相关性： 该提案的主要目的是开发下一代媒介，可以将治疗基因安全地递送给患有流血疾病的患者，称为血友病B。这些载体是从未知疾病的病毒中得出的，因此，这种病毒被发现，因此被期望更安全。因此，这种载体的发展是为了治疗和治愈血友病的，因此与公共卫生有关。