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Approach for in vivo gene delivery into hematopoietic stem cells for hemophilia A therapy

将基因体内递送至造血干细胞以治疗甲型血友病的方法

基本信息

批准号：
10162648
负责人：
ANDRE Michael LIEBER
金额：
$ 59.26万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-05-05 至 2023-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10162648
关键词：
AMD3100 Adenovirus Vector Adenoviruses Animal Model Animals Antibodies Apolipoprotein E Back Biomedical Engineering Blood Blood Cells Bone Marrow Bone Marrow Cells Bone Marrow Purging Bone Marrow Stem Cell CD34 gene CD46 Antigen CRISPR/Cas technology CSF3 gene Characteristics Chronic Hepatitis Clinical Clustered Regularly Interspaced Short Palindromic Repeats Communicable Diseases Complementary DNA Complex DNA DNA Repair Development Disadvantaged Erythrocytes Erythroid Cells Erythropoietin F8 gene Factor VIII Future Gene Amplification Gene Delivery Gene Transfer Gene-Modified Genes Genetic Genome Globin Goals Hematopoiesis Hematopoietic Stem Cell Mobilization Hematopoietic Stem Cell Transplantation Hematopoietic stem cells Hemophilia A Hepatocyte Hereditary Disease Home Human Hyperactivity In Vitro Injections Intervention Intravenous Kinetics Lentivirus Vector Life Liver Macaca Malignant Neoplasms Mammals Measures Mediating Modeling Modification Mus Outcome PTH gene Patients Pattern Production Protein Biosynthesis Proteins Protocols documentation Recombinant adeno-associated virus (rAAV)Regimen Risk Safety Sampling Serum Site Sleeping Beauty Stream System T-Lymphocyte Technology Testing Therapeutic Therapeutic Effect Therapeutic Monoclonal Antibodies Thymus Gland Time Transgenes Transgenic Organisms Transplantation Transposase Viral hepatitis Virus Inhibitors adenosine deaminase antibody inhibitor base cellular transduction clinical translation co-infection conditioning cost cost effectiveness cost efficient design efficacy study enzyme deficiency gene therapy genotoxicity homologous recombination hormone deficiency in vivo in vivo evaluation inhibitor/antagonist intravenous injection mouse model nonhuman primate novel strategies peripheral blood receptor safety study side effect stem cell gene therapy therapeutic protein therapeutic transgene transgene expression vector

项目摘要

ABSTRACT: We will test a new approach for the production of therapeutic proteins secreted from blood cells after in vivo gene delivery into hematopoietic stem cells (HSCs). This approach involves the mobilization of HSCs from the bone marrow followed by a single intravenous injection of integrating helper-dependent HDAd5/35++ adenovirus vectors. HSCs transduced in the peripheral blood return to the bone marrow where they persist long-term. Transgene integration is achieved either in a random pattern using a transposase or, in a site- specific pattern, through homology-directed DNA repair mechanisms. For a secreted transgene product, we will focus on human factor VIII expressed in erythrocytes after in vivo factor VIII gene transfer into HSCs. In contrast to currently used rAAV-mediated liver-directed hemophilia gene therapy, our technically simple and cost-efficient approach has the potential for a life-long cure with induction of tolerance to factor VIII. The Specific Aims are 1. Increase the efficacy and safety of transposase-based HDAd5/35++ in vivo HSC transduction through optimization of mobilization and vector injection regimens and through HSC in vivo expansion or selection mechanisms. 2. Test new HDAd5/35++ vector systems for targeted integration, including a vector that carries both a CRISPR-Cas9 to create site-specific DNA breaks and the homology template for integration. 3. Test the best in vivo HSC transduction system in a mouse model for hemophilia A. 4. Perform a pilot safety and efficacy study in non-human primates, which are the most adequate model for potential future studies in humans. The proposed 6-month study with repeated blood and bone marrow sampling will allow us to predict potential long-term side effects on hematopoiesis and follow the expansion of gene-edited HSCs over time.

抽象的：我们将测试一种在体内生产血细胞分泌的治疗性蛋白质的新方法将基因递送至造血干细胞 (HSC)。该方法涉及从 HSC 中调动骨髓，然后单次静脉注射整合辅助依赖性 HDAd5/35++ 腺病毒载体。外周血中转导的 HSC 返回骨髓并在那里持续存在长期。转基因整合可以使用转座酶以随机模式实现，也可以在位点上实现。特定模式，通过同源定向 DNA 修复机制。对于分泌型转基因产品，我们将重点关注体内因子 VIII 基因转移至 HSC 后红细胞中表达的人因子 VIII。在与目前使用的 rAAV 介导的肝定向血友病基因疗法相比，我们的技术简单且具有成本效益的方法有可能通过诱导对因子 VIII 的耐受而实现终生治愈。这具体目标是 1. 提高基于转座酶的 HDAd5/35++ 体内 HSC 的功效和安全性通过优化动员和载体注射方案以及通过体内 HSC 进行转导扩展或选择机制。 2. 测试新的HDAd5/35++矢量系统以进行有针对性的集成，包括携带 CRISPR-Cas9 以创建位点特异性 DNA 断裂和同源性的载体集成模板。 3. 在 A 型血友病小鼠模型中测试最佳的体内 HSC 转导系统。 4. 在非人类灵长类动物中进行试点安全性和有效性研究，这是最合适的模型未来潜在的人类研究。拟议的为期 6 个月的重复血液和骨髓研究采样将使我们能够预测对造血的潜在长期副作用，并跟踪随着时间的推移基因编辑的 HSC。