Hematopoietic gene therapy for hemophilia A

甲型血友病的造血基因治疗

• 批准号: 8230688
• 负责人: Christopher Bradley Doering
• 金额: $ 38.75万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8230688
• 关键词: A Mouse; Affect; Anabolism; Antibodies; Biology; Biomedical Engineering; Blood; Blood Cells; Blood Clot; Blood Coagulation Factor; Blood coagulation; Bone Marrow Transplantation; CD34 gene; Cell Lineage; Cells; Clinical; Clinical Trials; Data; Development; Disease; Drug Kinetics; Engineering; Engraftment; Factor VIII; Family suidae; Gene Mutation; Gene Transfer; Genetic; Goals; HIV; Hematopoietic; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Hemophilia A; Hemorrhage; Human; Immune Tolerance; Immune response; Immunization; Immunologic Deficiency Syndromes; Immunosuppression; In Vitro; Individual; Insertional Mutagenesis; Intravenous infusion procedures; Joints; Lentivirus Vector; Mesenchymal Stem Cells; Methods; Modeling; Modification; Morbidity - disease rate; Mus; Outcome; Pathway interactions; Patients; Performance; Phase I Clinical Trials; Plasma Proteins; Population; Procedures; Production; Property; Proteins; Protocols documentation; Reagent; Recombinants; Regimen; Reporting; Retroviral Vector; Retroviridae; SIV; Safety; Stem cell transplant; Stem cells; System; Testing; Therapeutic; Toxic effect; Transgenes; Transgenic Organisms; Translating; Transplantation; Transplantation Conditioning; Treatment Efficacy; Viral Genes; Xenograft procedure; arthropathies; base; clinically relevant; clinically significant; conditioning; cost; design; expression vector; gene therapy; gene therapy clinical trial; gene transfer vector; genetically modified cells; human F8 protein; in vivo; inhibitor/antagonist; mouse model; novel; preclinical study; promoter; protein expression; public health relevance; simian human immunodeficiency virus; stem; stem cell biology; success

DESCRIPTION (provided by applicant): Hemophilia A is a congenital bleeding disorder caused by genetic mutations affecting a plasma protein, termed factor VIII (fVIII), whose function is to facilitate blood clotting. State of the art treatment for hemophilia A consists of frequent intravenous infusions of fVIII containing products. The current limitations to treating hemophilia are 1) the cost of fVIII products, 3) the development of immune responses against fVIII that block treatment efficacy, 3) morbidity due to joint disease resulting from repeated bleeding into individual joints and 4) the limitation of treatment to 30% of the world population. Due to the limited amount of fVIII needed to provide clinical benefit to the patient, hemophilia A is an attractive disease target for gene therapy, and three phase 1 clinical trials have been conducted. The outcome of these trials has been disappointing due to the extremely low, non-therapeutic levels of fVIII produced in each of the gene therapy strategies. We recently showed that a modified porcine fVIII transgene, designated BDDpfVIII, facilitates very high-level protein expression, and we demonstrated proof-of-concept that this transgene functions extremely efficiently in a mouse model of hemophilia A following transplantation of genetically-modified hematopoietic stem cells (HSCs). Specifically, we have shown that the expression of BDDpfVIII is superior to other bioengineered human fVIII expression constructs and that genetic modification and transplantation of HSCs results in curative fVIII levels. Additionally, curative fVIII activity levels are achieved after transplantation of BDDpfVIII-transduced HSCs following low-toxicity pre-transplantation conditioning with targeted immunosuppression, even in the context of pre-existing anti-human fVIII inhibitors. Therefore, we have overcome the major hurdle of low-level expression using a transgene that encodes a protein that has been used successfully in patients with hemophilia A. In the current application, we propose to more fully characterize the use of the high-expression construct and further our understanding of the critical parameters involved with this novel gene therapy strategy and study the biology of non-physiological BDDpfVIII expression in hematopoietic (blood) cells. To advance our studies toward clinical significance, we propose to 1) test clinically relevant HSC transplant conditioning regimens that more closely resemble those used routinely in human bone marrow transplant protocols and 2) test recombinant lentiviral vectors that have been demonstrated to display a reduction of insertional mutagenesis compared to oncoretroviruses. Finally, the optimized lentiviral vector(s) encoding BDDpfVIII will be tested for the ability to genetically modify human HSCs. PUBLIC HEALTH RELEVANCE: Hemophilia A is a bleeding disorder caused by insufficiency of a blood clotting factor, designated factor VIII, for which gene therapy offers a potential cure. However, pre-clinical studies and clinical trials showed that a major limitation to a successful gene therapy treatment is the extremely poor expression of fVIII from the human fVIII transgene, which we recently overcame by introducing a porcine fVIII transgene. We demonstrated that use of the porcine transgene results in up to 100-fold greater fVIII production than the human version, and more recently, we demonstrated that a similar differential is observed following gene transfer into blood stem cells using recombinant retroviruses. Our current studies focus on better understanding the critical pharmacologic parameters involved in generating curative fVIII levels with this procedure using a mouse model of hemophilia A with the goal of understanding how this application can best be translated to clinical successes.

描述（由申请人提供）：A 型血友病是一种先天性出血性疾病，由影响血浆蛋白（称为因子 VIII (fVIII)）的基因突变引起，其功能是促进血液凝固。 A 型血友病的最先进治疗方法包括频繁静脉输注含有 fVIII 的产品。目前治疗血友病的局限性包括 1) fVIII 产品的成本，3) 针对 fVIII 的免疫反应的发展阻碍了治疗效果，3) 由于个别关节反复出血导致的关节疾病导致的发病率，以及 4) 治疗仅限于世界人口的 30%。由于为患者提供临床益处所需的 fVIII 量有限，因此血友病 A 是基因治疗的一个有吸引力的疾病靶标，并且已经进行了三项 1 期临床试验。由于每种基因治疗策略中产生的 fVIII 水平极低且非治疗水平，这些试验的结果令人失望。我们最近证明，一种被命名为 BDDpfVIII 的改良猪 fVIII 转基因可促进非常高水平的蛋白质表达，并且我们证明了这种转基因在移植转基因造血干细胞 (HSC) 后的 A 型血友病小鼠模型中极其有效地发挥作用。具体来说，我们已经证明 BDDpfVIII 的表达优于其他生物工程化的人类 fVIII 表达构建体，并且 HSC 的基因修饰和移植可达到治疗性的 fVIII 水平。此外，即使在预先存在的抗人 fVIII 抑制剂的情况下，在低毒性移植前调理和靶向免疫抑制后，BDDpfVIII 转导的 HSC 移植后也能达到治疗性 fVIII 活性水平。因此，我们使用编码已成功用于血友病A患者的蛋白质的转基因克服了低水平表达的主要障碍。在当前的应用中，我们建议更全面地表征高表达构建体的使用，并进一步了解与这种新型基因治疗策略相关的关键参数，并研究造血（血液）细胞中非生理性BDDpfVIII表达的生物学。为了推进我们的研究具有临床意义，我们建议 1) 测试临床相关的 HSC 移植预处理方案，该方案更类似于人类骨髓移植方案中常规使用的方案，2) 测试重组慢病毒载体，与致癌逆转录病毒相比，该载体已被证明显示插入诱变减少。最后，将测试编码 BDDpfVIII 的优化慢病毒载体对人类 HSC 进行基因修饰的能力。 公众健康相关性：A 型血友病是一种由凝血因子（称为因子 VIII）不足引起的出血性疾病，基因治疗提供了潜在的治愈方法。然而，临床前研究和临床试验表明，成功的基因治疗的一个主要限制是人类 fVIII 转基因中 fVIII 的表达极差，我们最近通过引入猪 fVIII 转基因克服了这一问题。我们证明使用猪转基因可产生比人类转基因高 100 倍的 fVIII 产量，最近，我们证明使用重组逆转录病毒将基因转移到血液干细胞后观察到类似的差异。我们目前的研究重点是更好地了解使用 A 型血友病小鼠模型通过该程序产生治疗性 fVIII 水平所涉及的关键药理学参数，目的是了解如何将该应用最好地转化为临床成功。