Hematopoietic gene therapy for hemophilia A

描述（由申请人提供）：血友病A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。血友病A的最新治疗包括频繁静脉输注含FVIII的产品。目前治疗血友病的局限性是1）fVIII产品的成本，3）针对fVIII的免疫应答的发展阻碍了治疗疗效，3）由于单个关节反复出血导致的关节疾病导致的发病率和4）治疗仅限于30%的世界人口。由于为患者提供临床获益所需的fVIII量有限，血友病A是基因治疗的一个有吸引力的疾病靶点，已经进行了三项1期临床试验。这些试验的结果令人失望，因为在每种基因治疗策略中产生的fVIII水平极低，非治疗性。我们最近表明，一种修饰的猪fVIII转基因，命名为BDDpfVIII，促进非常高水平的蛋白质表达，我们证明了概念验证，这种转基因功能非常有效地在小鼠模型血友病A移植后的遗传修饰的造血干细胞（HSC）。具体地，我们已经表明，BDDpfVIII的表达上级其他生物工程化的人fVIII表达构建体，并且HSC的遗传修饰和移植导致治愈性fVIII水平。此外，即使在预先存在的抗人fVIII抑制剂的情况下，在低毒性移植前预处理和靶向免疫抑制后，BDDpfVIII转导的HSC移植后也可达到治愈性fVIII活性水平。因此，我们已经克服了低水平表达的主要障碍，使用了一种编码蛋白质的转基因，该蛋白质已成功用于血友病A患者。在本申请中，我们建议更充分地表征高表达构建体的使用，并进一步理解与这种新的基因治疗策略相关的关键参数，并研究造血（血）细胞中非生理性BDDpfVIII表达的生物学。为了使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，这些方案更接近于人骨髓移植方案中常规使用的方案，2）测试重组慢病毒载体，这些载体已被证明与肿瘤逆转录病毒相比显示插入突变减少。最后，将测试编码BDDpfVIII的优化的慢病毒载体遗传修饰人HSC的能力。 公共卫生相关性：血友病A是一种出血性疾病，由血液凝固因子不足引起，命名为因子VIII，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，一个成功的基因疗法治疗的主要限制是从人的FVIII转基因，我们最近通过引入猪的FVIII转基因克服了FVIII的表达非常差。我们证明，使用猪转基因的结果高达100倍以上的FVIII生产比人类版本，最近，我们证明了类似的差异，观察到基因转移到血液干细胞使用重组逆转录病毒。我们目前的研究重点是更好地了解使用血友病A小鼠模型通过该程序产生治愈性fVIII水平所涉及的关键药理学参数，目的是了解该应用如何最好地转化为临床成功。

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.